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Lentiviral transduction of miR-371~373 cluster restores miR-371~373 expression in different SCs

Antibodies, primers, and lentiviral particles (S1); in vivo tumorigenicity for SW480 vs. SW620 (S2); top 20 differentially expressed miRNAs (S3); peritoneal cancer index scores (S4)

The vast majority of colorectal cancer–related deaths can be attributed to metastatic spreading of the disease. Therefore, deciphering molecular mechanisms of metastatic dissemination is a key prerequisite to improve future treatment options. With this aim, we took advantage of different colorectal cancer cell lines and recently established primary cultures enriched in colon cancer stem cells, also known as tumor-initiating cells (TIC), to identify genes and miRNAs with regulatory functions in colorectal cancer progression. We show here that metastasis-derived TICs display increased capacity for self-renewal, TGFβ signaling activity, and reduced expression of the miR-371∼373 cluster compared with nonmetastatic cultures. TGFβ receptor 2 (TGFBR2) and aldehyde dehydrogenase A1 (ALDH1A1) were identified as important target genes of the miR-371∼373 cluster. In addition, TGFBR2 repression, either by direct knockdown or indirectly via overexpression of the entire miR-371∼373 cluster, decreased tumor-initiating potential of TICs. We observed significantly reduced in vitro self-renewal activity as well as lowered tumor initiation and metastatic outgrowth capacity in vivo following stable overexpression of the miR-371∼373 cluster in different colon TIC cultures. Inhibitor of DNA binding 1 (ID1) was affected by both TGFBR2 and miR-371∼373 cluster alterations. Functional sphere and tumor formation as well as metastatic dissemination assays validated the link between miR-371∼373 and ID1. Altogether, our results establish the miR-371∼373/TGFBR2/ID1 signaling axis as a novel regulatory mechanism of TIC self-renewal and metastatic colonization.Significance: These findings establish the miR-371∼373/TGFBR2/ID1 signaling axis as a novel mechanism regulating self-renewal of tumor-initiating cell and metastatic colonization, potentially opening new concepts for therapeutic targeting of cancer metastasis.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/14/3793/F1.large.jpg. Cancer Res; 78(14); 3793–808. ©2018 AACR.

Midbrain dopaminergic neurons (mDANs) control voluntary movement, cognition, and reward behavior under physiological conditions and are implicated in human diseases such as Parkinson’s disease (PD). Many transcription factors (TFs) controlling human mDAN differentiation during development have been described, but much of the regulatory landscape remains undefined. Using a tyrosine hydroxylase (TH) iPSC reporter line, we have generated time series transcriptomic and epigenomic profiles of purified mDANs during differentiation. Integrative analysis predicted novel central regulators of mDAN differentiation and super-enhancers were used to prioritize key TFs. We find LBX1, NHLH1 and NR2F1/2 to be necessary for mDAN differentiation and show that overexpression of either LBX1 or NHLH1 can also improve mDAN specification. NHLH1 is necessary for the induction of neuronal miR-124, while LBX1 regulates cholesterol biosynthesis, possibly through mTOR signaling. Consistently, rapamycin treatment led to an inhibition of mDAN differentiation. Thus, our work reveals novel regulators of human mDAN differentiation.

Cytokines orchestrate responses to pathogens and in inflammatory processes, but they also play an important role in cancer by shaping the expression levels of cytokine response genes. Here, we conducted a large profiling study comparing miRNome and mRNA transcriptome data generated following different cytokine stimulations. Transcriptomic responses to STAT1- (IFNγ, IL-27) and STAT3-activating cytokines (IL6, OSM) were systematically compared in nine cancerous and non-neoplastic cell lines of different tissue origins (skin, liver and colon). The largest variation in our datasets was seen between cell lines of the three different tissues rather than stimuli. Notably, the variability in miRNome datasets was a lot more pronounced than in mRNA data. Our data also revealed that cells of skin, liver and colon tissues respond very differently to cytokines and that the cell signaling networks activated or silenced in response to STAT1- or STAT3-activating cytokines are specific to the tissue and the type of cytokine. However, globally, STAT1-activating cytokines had stronger effects than STAT3-inducing cytokines with most significant responses in liver cells, showing more genes upregulated and with higher fold change. A more detailed analysis of gene regulations upon cytokine stimulation in these cells provided insights into STAT1- versus STAT3-driven processes in hepatocarcinogenesis. Finally, independent component analysis revealed interconnected transcriptional networks distinct between cancer cells and their healthy counterparts.

The NF-κB signaling pathway is crucial during development and inflammatory processes. We have previously shown that NF-κB activation induces dedifferentiation of astrocytes into neural progenitor cells (NPCs). Here, we provide evidence&nbsp, that the NF-κB pathway plays also a fundamental role during the differentiation of NPCs into astrocytes. First, we show that the NF-κB pathway is essential to initiate astrocytic differentiation as its early inhibition induces NPC apoptosis and impedes their differentiation. Second, we demonstrate that persistent NF-κB activation affects NPC-derived astrocyte differentiation. Tumor necrosis factor (TNF)-treated NPCs show NF-κB activation, maintain their multipotential and proliferation properties, display persistent expression of immature markers and inhibit astrocyte markers. Third, we analyze the effect of &nbsp, NF-κB activation on the main known astrocytic differentiation pathways, such as NOTCH and JAK-STAT. Our findings suggest that the NF-κB pathway plays a dual fundamental role during NPC differentiation into astrocytes: it promotes astrocyte specification, but its persistent activation impedes their differentiation.

Repeated systemic challenge with lipopolysaccharides (LPS) can induce microglia activation and inflammatory neurodegeneration in the substantia nigra pars compacta region of mice. We now explored the role of mononuclear phagocytes associated nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX‐2) in inflammatory neurodegeneration. Cybb ‐deficient NOX‐2 knock‐out (KO) and control wild type (WT) mice were treated intraperitoneally daily over four consecutive days with 1 μg/gbw/day LPS. Transcriptome analysis by RNA‐seq of total brain tissue indicated increased LPS‐induced upregulation of genes belonging to the reactive oxygen species and reactive nitrogen species production, complement and lysosome activation as well as apoptosis and necroptosis in WT compared to NOX‐2 KO mice. Validation of up‐regulated gene transcripts via qRT‐PCR confirmed that LPS‐challenged NOX‐2 KO mice expressed lower levels of the microglial phagocytosis‐related genes Nos2 , Cd68 , Aif1/Iba1 , Cyba , Itgam , and Fcer1g compared to WT mice at Day 5 after systemic inflammatory challenge, but no significant differences in the pro‐inflammatory genes Tnfα and Il1b as well as microglial IBA1 and CD68 intensities were observed between both genotypes. Furthermore, loss of tyrosine hydroxylase positive (TH+) and NeuN positive neurons in the substantia nigra pars compacta upon repeated systemic LPS application were attenuated in NOX‐2 KO mice. Thus, our data demonstrate that loss of dopaminergic neurons in the substantia nigra pars compacta after repeated systemic challenge with LPS is associated with a microglial phagocytosis‐related gene activation profile involving the NADPH oxidase subunit Cybb/gp91phox.

Temporal data on gene expression and context-specific open chromatin states can improve identification of key transcription factors (TFs) and the gene regulatory networks (GRNs) controlling cellular differentiation. However, their integration remains challenging. Here, we delineate a general approach for data-driven and unbiased identification of key TFs and dynamic GRNs, called EPIC-DREM. We generated time-series transcriptomic and epigenomic profiles during differentiation of mouse multipotent bone marrow stromal cell line (ST2) toward adipocytes and osteoblasts. Using our novel approach we constructed time-resolved GRNs for both lineages and identifed the shared TFs involved in both differentiation processes. To take an alternative approach to prioritize the identified shared regulators, we mapped dynamic super-enhancers in both lineages and associated them to target genes with correlated expression profiles. The combination of the two approaches identified aryl hydrocarbon receptor (AHR) and Glis family zinc finger 1 (GLIS1) as mesenchymal key TFs controlled by dynamic cell type-specific super-enhancers that become repressed in both lineages. AHR and GLIS1 control differentiation-induced genes and their overexpression can inhibit the lineage commitment of the multipotent bone marrow-derived ST2 cells.

Interleukin-6 (IL-6)-type cytokines play important roles in liver (patho-)biology. For instance, they regulate the acute phase response to inflammatory signals and are involved in hepatocarcinogenesis. Much is known about the regulation of protein-coding genes by cytokines whereas their effects on the miRNome is less well understood. We performed a microarray screen to identify microRNAs (miRNAs) in human hepatocytes which are modulated by IL-6-type cytokines. Using samples of 2 donors, 27 and 68 miRNAs (out of 1,733) were found to be differentially expressed upon stimulation with hyper-IL-6 (HIL-6) for up to 72 h, with an overlap of 15 commonly regulated miRNAs. qPCR validation revealed that miR-146b-5p was also consistently up-regulated in hepatocytes derived from 2 other donors. Interestingly, miR-146b-5p (but not miR-146a-5p) was induced by IL-6-type cytokines (HIL-6 and OSM) in non-transformed liver-derived PH5CH8 and THLE2 cells and in Huh-7 hepatoma cells, but not in HepG2 or Hep3B hepatoma cells. We did not find evidence for a differential regulation of miR-146b-5p expression by promoter methylation, also when analyzing the TCGA data set on liver cancer samples. Inducible overexpression of miR-146b-5p in PH5CH8 cells followed by RNA-Seq analysis revealed effects on multiple mRNAs, including those encoding IRAK1 and TRAF6 crucial for Toll-like receptor signaling. Indeed, LPS-mediated signaling was attenuated upon overexpression of miR-146b-5p, suggesting a regulatory loop to modulate inflammatory signaling in hepatocytes. Further validation experiments suggest DNAJC6, MAGEE1, MPHOSPH6, PPP2R1B, SLC10A3, SNRNP27, and TIMM17B to be novel targets for miR-146b-5p (and miR-146a-5p). IL-6-type cytokines up-regulate miR-146b-5p expression in liver cells, an anti-inflammatory miRNA with multiple targets shared with miR-146a-5p.

Interleukin-27 (IL27) is a type-I-cytokine of the IL6/IL12 family predominantly secreted by activated macrophages and dendritic cells. In the liver, IL27 expression was observed to be upregulated in patients with hepatitis B, and sera of hepatocellular carcinoma (HCC) patients contain significantly elevated levels of IL27 compared to healthy controls or patients with hepatitis and/or liver cirrhosis. In this study, we show that IL27 induces STAT1 and STAT3 phosphorylation in 5 HCC lines and 3 different types of non-transformed liver cells. We were especially interested in the relevance of the IL27-induced STAT3 activation in liver cells. Thus, we compared the IL27 responses with those induced by IFNγ (STAT1-dominated response) or IL6-type cytokines (IL6, hyper-IL6 (hy-IL6) or OSM) (STAT3-dominated response) by microarray analysis and find that in HCC cells, IL27 induces an IFNγ-like, STAT1-dependent transcriptional response, but we do not find an effective STAT3-dependent response. Validation experiments corroborate the finding from the microarray evaluation. Interestingly, the availability of STAT1 seems critical in the shaping of the IL27 response, as the siRNA knock-down of STAT1 revealed the ability of IL27 to induce the acute-phase protein γ-fibrinogen, a typical IL6 family characteristic. Moreover, we describe a crosstalk between the signaling of IL6-type cytokines and IL27: responses to the gp130-engaging cytokine IL27 (but not those to IFNs) can be inhibited by IL6-type cytokine pre-stimulation, likely by a SOCS3-mediated mechanism. Thus, IL27 recapitulates IFNγ responses in liver cells, but differs from IFNγ by its sensitivity to SOCS3 inhibition.

The microglial triggering receptor expressed on myeloid cells 2 (TREM2) signals via the activatory membrane adaptor molecule TYROBP. Genetic variants or mutations of TREM2 or TYROBP have been linked to inflammatory neurodegenerative diseases associated with aging. The typical aging process goes along with microglial changes and mild neuronal loss, but the exact contribution of TREM2 is still unclear. Aged TREM2 knock-out mice showed decreased age-related neuronal loss in the substantia nigra and the hippocampus. Transcriptomic analysis of the brains of 24 months old TREM2 knock-out mice revealed 211 differentially expressed genes mostly downregulated and associated with complement activation and oxidative stress response pathways. Consistently, 24 months old TREM2 knock-out mice showed lower transcription of microglial (Aif1 and Tmem119), oxidative stress markers (Inos, Cyba, and Cybb) and complement components (C1qa, C1qb, C1qc, C3, C4b, Itgam, and Itgb2), decreased microglial numbers and expression of the microglial activation marker Cd68, as well as accumulation of oxidized lipids. Cultured microglia of TREM2 knock-out mice showed reduced phagocytosis and oxidative burst. Thus, microglial TREM2 contributes to age-related microglial changes, phagocytic oxidative burst, and loss of neurons with possible detrimental effects during physiological aging.

Aim: Prediction of genes under dynamic post-transcriptional regulation from epigenomic data. Materials & methods: We used time-series profiles of chromatin immunoprecipitation-seq data of histone modifications from differentiation of mesenchymal progenitor cells toward adipocytes and osteoblasts to predict gene expression levels at five time points in both lineages and estimated the deviation of those predictions from the RNA-seq measured expression levels using linear regression. Results & conclusion: The genes with biggest changes in their estimated stability across the time series are enriched for noncoding RNAs and lineage-specific biological processes. Clustering mRNAs according to their stability dynamics allows identification of post-transcriptionally coregulated mRNAs and their shared regulators through sequence enrichment analysis. We identify miR-204 as an early induced adipogenic microRNA targeting Akr1c14 and Il1rl1.

The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixture events for extinct taxa1. However, the irreversible post-mortem degradation2 of ancient DNA has so far limited its recovery—outside permafrost areas—to specimens that are not older than approximately 0.5 million years (Myr)3. By contrast, tandem mass spectrometry has enabled the sequencing of approximately 1.5-Myr-old collagen type I4, and suggested the presence of protein residues in fossils of the Cretaceous period5—although with limited phylogenetic use6. In the absence of molecular evidence, the speciation of several extinct species of the Early and Middle Pleistocene epoch remains contentious. Here we address the phylogenetic relationships of the Eurasian Rhinocerotidae of the Pleistocene epoch7–9, using the proteome of dental enamel from a Stephanorhinus tooth that is approximately 1.77-Myr old, recovered from the archaeological site of Dmanisi (South Caucasus, Georgia)10. Molecular phylogenetic analyses place this Stephanorhinus as a sister group to the clade formed by the woolly rhinoceros (Coelodonta antiquitatis) and Merck’s rhinoceros (Stephanorhinus kirchbergensis). We show that Coelodonta evolved from an early Stephanorhinus lineage, and that this latter genus includes at least two distinct evolutionary lines. The genus Stephanorhinus is therefore currently paraphyletic, and its systematic revision is needed. We demonstrate that sequencing the proteome of Early Pleistocene dental enamel overcomes the limitations of phylogenetic inference based on ancient collagen or DNA. Our approach also provides additional information about the sex and taxonomic assignment of other specimens from Dmanisi. Our findings reveal that proteomic investigation of ancient dental enamel—which is the hardest tissue in vertebrates11, and is highly abundant in the fossil record—can push the reconstruction of molecular evolution further back into the Early Pleistocene epoch, beyond the currently known limits of ancient DNA preservation. Palaeoproteomic analysis of dental enamel from an Early Pleistocene Stephanorhinus resolves the phylogeny of Eurasian Rhinocerotidae, by enabling the reconstruction of molecular evolution beyond the limits of ancient DNA preservation.

// Pit Ullmann 1 , Komal Qureshi-Baig 1 , Fabien Rodriguez 1 , Aurelien Ginolhac 1 , Yannic Nonnenmacher 2 , Dominik Ternes 1 , Jil Weiler 1 , Karoline Gabler 1 , Christelle Bahlawane 1 , Karsten Hiller 2 , Serge Haan 1 , Elisabeth Letellier 1 1 Life Sciences Research Unit, University of Luxembourg, L-4367 Belvaux, Luxembourg 2 Luxembourg Centre for Systems Biomedicine, L-4367 Belvaux, Luxembourg Correspondence to: Elisabeth Letellier, email: elisabeth.letellier@uni.lu Keywords: colorectal cancer, tumor-initiating cell, hypoxia, miR-210, self-renewal capacity Received: May 20, 2016 Accepted: August 25, 2016 Published: August 31, 2016 ABSTRACT Low oxygen concentrations (hypoxia) are known to affect the cellular metabolism and have been suggested to regulate a subpopulation of cancer cells with tumorigenic properties, the so-called tumor-initiating cells (TICs). To better understand the mechanism of hypoxia-induced TIC activation, we set out to study the role of hypoxia-responsive miRNAs in recently established colon cancer patient-derived TICs. We were able to show that low oxygen concentrations consistently lead to the upregulation of miR-210 in different primary TIC-enriched cultures. Both stable overexpression of miR-210 and knockdown of its target gene ISCU resulted in enhanced TIC self-renewal. We could validate the tumorigenic properties of miR- 210 in in vivo experiments by showing that ectopic expression of miR-210 results in increased tumor incidence. Furthermore, enhanced miR-210 expression correlated with reduced TCA cycle activity and increased lactate levels. Importantly, by blocking lactate production via inhibition of LDHA, we could reverse the promoting effect of miR-210 on self-renewal capacity, thereby emphasizing the regulatory impact of the glycolytic phenotype on colon TIC properties. Finally, by assessing expression levels in patient tissue, we could demonstrate the clinical relevance of the miR-210/ISCU signaling axis for colorectal carcinoma. Taken together, our study highlights the importance of hypoxia-induced miR-210 in the regulation of colon cancer initiation.

Accumulating evidence suggests that dysbiosis, a state of pathologic imbalance in the human gut microbiome, is present in patients suffering from colorectal cancer (CRC). Several microbiome studies identified specific bacteria that are associated with CRC, among which Fusobacteria were shown to directly interact with cancer or immune cells of their host. However, only a limited number of CRC-associated microbes have been studied for host-microbial interactions; hence, the role of bacteria in the etiology of the disease remains unknown. Accordingly, our work aims at the development of a methodologic workflow for studying CRC-associated bacteria and their role in colon cancer tumor initiation and progression. In a first step, we identified CRC-associated bacteria that are enriched at the tumor site of CRC patients. Therefore, we used publicly available datasets and an in-house patient sample collection. Then, we predicted and optimized bacterial growth in silico by using a genome-scale metabolic reconstruction model combined with a constraint-based modeling approach. Finally, we implemented CRC-associated bacteria together with established primary CRC patient cultures into the microfluidics-based human-microbial crosstalk model (HuMiX). Our workflow allowed to analyze host-microbial interaction mechanisms of CRC-associated bacteria on a transcriptomic, proteomic, and metabolomic level. Citation Format: Dominik Ternes, Martine Schmitz, Léa Grandmougin, Mina Tsenkova, Eric Koncina, Aurélien Ginolhac, Jessica Karta, Diana Kuhn, Javier Ramiro Garcia, Kacy Greenhalgh, Paul Wilmes, Elisabeth Letellier, Serge Haan. Understanding the role of colorectal cancer-associated microbes in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr A09.

The microglial triggering receptor expressed on myeloid cells 2 (TREM2) signals via the activatory membrane adaptor molecule TYROBP. Genetic variants or mutations of TREM2 or TYROBP have been linked to inflammatory neurodegenerative diseases associated with aging. The typical aging process goes along with microglial changes and mild neuronal loss, but the exact contribution of TREM2 is still unclear. Aged TREM2 knock‐out mice showed decreased age‐related neuronal loss in the substantia nigra and the hippocampus. Transcriptomic analysis of the brains of 24 months old TREM2 knock‐out mice revealed 211 differentially expressed genes mostly downregulated and associated with complement activation and oxidative stress response pathways. Consistently, 24 months old TREM2 knock‐out mice showed lower transcription of microglial (Aif1 and Tmem119), oxidative stress markers (Inos, Cyba, and Cybb) and complement components (C1qa, C1qb, C1qc, C3, C4b, Itgam, and Itgb2), decreased microglial numbers and expression of the microglial activation marker Cd68, as well as accumulation of oxidized lipids. Cultured microglia of TREM2 knock‐out mice showed reduced phagocytosis and oxidative burst. Thus, microglial TREM2 contributes to age‐related microglial changes, phagocytic oxidative burst, and loss of neurons with possible detrimental effects during physiological aging.

Interleukin-27 (IL27) is a type-I cytokine of the IL6/IL12 family and is predominantly secreted by activated macrophages and dendritic cells. We show that IL27 induces STAT factor phosphorylation in cancerous cell lines of different tissue origin. IL27 leads to STAT1 phosphorylation and recapitulates an IFN-γ-like response in the microarray analyses, with up-regulation of genes involved in antiviral defense, antigen presentation, and immune suppression. Like IFN-γ, IL27 leads to an up-regulation of TAP2 and MHC-I proteins, which mediate increased tumor immune clearance. However, both cytokines also upregulate proteins such as PD-L1 (CD274) and IDO-1, which are associated with immune escape of cancer. Interestingly, differential expression of these genes was observed within the different cell lines and when comparing IL27 to IFN-γ. In coculture experiments of hepatocellular carcinoma (HCC) cells with peripheral blood mononuclear cells, pre-treatment of the HCC cells with IL27 resulted in lowered IL2 production by anti-CD3/-CD28 activated T-lymphocytes. Addition of anti-PD-L1 antibody, however, restored IL2 secretion. The levels of other TH1 cytokines were also enhanced or restored upon administration of anti-PD-L1. In addition, we show that the suppression of IL27 signaling by IL6-type cytokine pre-stimulation—mimicking a situation occurring, for example, in IL6-secreting tumors or in tumor inflammation–induced cachexia—can be antagonized by antibodies against IL6-type cytokines or their receptors. Therapeutically, the antitumor effects of IL27 (mediated, e.g., by increased antigen presentation) might thus be increased by combining IL27 with blocking antibodies against PD-L1 or/and IL6-type cytokines. Anti-IL6 or anti-PD-L1 influence IL27 signaling effects in HCC cells by preventing IL6 mediated inhibition of IL27 or by mediating an increase in immunogenicity, respectively.

Ancient DNA (aDNA) sequencing has enabled unprecedented reconstruction of speciation, migration, and admixture events for extinct taxa1. Outside the permafrost, however, irreversible aDNA post-mortem degradation2 has so far limited aDNA recovery within the ˜0.5 million years (Ma) time range3. Tandem mass spectrometry (MS)-based collagen type I (COL1) sequencing provides direct access to older biomolecular information4, though with limited phylogenetic use. In the absence of molecular evidence, the speciation of several Early and Middle Pleistocene extinct species remain contentious. In this study, we address the phylogenetic relationships of the Eurasian Pleistocene Rhinocerotidae5-7 using ˜1.77 million years (Ma) old dental enamel proteome sequences of a Stephanorhinus specimen from the Dmanisi archaeological site in Georgia (South Caucasus)8. Molecular phylogenetic analyses place the Dmanisi Stephanorhinus as a sister group to the woolly (Coelodonta antiquitatis) and Merck’s rhinoceros (S. kirchbergensis) clade. We show that Coelodonta evolved from an early Stephanorhinus lineage and that this genus includes at least two distinct evolutionary lines. As such, the genus Stephanorhinus is currently paraphyletic and its systematic revision is therefore needed. We demonstrate that Early Pleistocene dental enamel proteome sequencing overcomes the limits of ancient collagen- and aDNA-based phylogenetic inference, and also provides additional information about the sex and taxonomic assignment of the specimens analysed. Dental enamel, the hardest tissue in vertebrates, is highly abundant in the fossil record. Our findings reveal that palaeoproteomic investigation of this material can push biomolecular investigation further back into the Early Pleistocene.

An underlying endothelial dysfunction plays a fundamental role in the pathogenesis of cardiovascular events and is the central feature of atherosclerosis. The protein-based communication between leukocytes and inflamed endothelial cells leading to diapedesis has been largely investigated and several key players such as IL6, TNFα, or the damage associated molecular pattern molecule (DAMP) calprotectin are now well identified. However, regarding cytokine IL27, the controversial current knowledge about its inflammatory role and the involved regulatory elements requires clarification. Therefore, we examined the inflammatory impact of IL27 on primary endothelial cells and the potentially modulatory effect of calprotectin on both transcriptome and proteome levels. A qPCR-based screening demonstrated high IL27-mediated gene expression ofIL7,IL15,CXCL10, andCXCL11. Calprotectin time-dependent downregulatory effects were observed on IL27-inducedIL15andCXCL10gene expression. A mass spectrometry-based approach of IL27 ± calprotectin cell stimulation enlightened a calprotectin modulatory role in the expression of 28 proteins, mostly involved in the mechanism of leukocyte transmigration. Furthermore, we showed evidence for STAT1 involvement in this process. Our findings provide new evidence about the IL27-dependent proinflammatory signaling which may be under the control of calprotectin and highlight the need for further investigations on molecules which might have antiatherosclerotic functions.

Background: Selecting the most beneficial treatment regimens for colorectal cancer (CRC) patients remains challenging due to a lack of prognostic markers. Members of the Myosin family, proteins recognised to have a major role in trafficking and polarisation of cells, have recently been reported to be closely associated with several types of cancer and might thus serve as potential prognostic markers in the context of CRC. Methods: We used a previously established meta-analysis of publicly available gene expression data to analyse the expression of different members of the Myosin V family, namely MYO5A, 5B, and 5C, in CRC. Using laser-microdissected material as well as tissue microarrays from paired human CRC samples, we validated both RNA and protein expression of Myosin Vb (MYO5B) and its known adapter proteins (RAB8A and RAB25) in an independent patient cohort. Finally, we assessed the prognostic value of both MYO5B and its adapter-coupled combinatorial gene expression signatures. Results: The meta-analysis as well as an independent patient cohort study revealed a methylation-independent loss of MYO5B expression in CRC that matched disease progression. Although MYO5B mutations were identified in a small number of patients, these cannot be solely responsible for the common downregulation observed in CRC patients. Significantly, CRC patients with low MYO5B expression displayed shorter overall, disease-, and metastasis-free survival, a trend that was further reinforced when RAB8A expression was also taken into account. Conclusions: Our data identify MYO5B as a powerful prognostic biomarker in CRC, especially in early stages (stages I and II), which might help stratifying patients with stage II for adjuvant chemotherapy.

The vast majority of colorectal cancer–related deaths can be attributed to metastatic spreading of the disease. Therefore, deciphering molecular mechanisms of metastatic dissemination is a key prerequisite to improve future treatment options. With this aim, we took advantage of different colorectal cancer cell lines and recently established primary cultures enriched in colon cancer stem cells, also known as tumor-initiating cells (TIC), to identify genes and miRNAs with regulatory functions in colorectal cancer progression. We show here that metastasis-derived TICs display increased capacity for self-renewal, TGFβ signaling activity, and reduced expression of the miR-371∼373 cluster compared with nonmetastatic cultures. TGFβ receptor 2 (TGFBR2) and aldehyde dehydrogenase A1 (ALDH1A1) were identified as important target genes of the miR-371∼373 cluster. In addition, TGFBR2 repression, either by direct knockdown or indirectly via overexpression of the entire miR-371∼373 cluster, decreased tumor-initiating potential of TICs. We observed significantly reduced in vitro self-renewal activity as well as lowered tumor initiation and metastatic outgrowth capacity in vivo following stable overexpression of the miR-371∼373 cluster in different colon TIC cultures. Inhibitor of DNA binding 1 (ID1) was affected by both TGFBR2 and miR-371∼373 cluster alterations. Functional sphere and tumor formation as well as metastatic dissemination assays validated the link between miR-371∼373 and ID1. Altogether, our results establish the miR-371∼373/TGFBR2/ID1 signaling axis as a novel regulatory mechanism of TIC self-renewal and metastatic colonization. Significance: These findings establish the miR-371∼373/TGFBR2/ID1 signaling axis as a novel mechanism regulating self-renewal of tumor-initiating cell and metastatic colonization, potentially opening new concepts for therapeutic targeting of cancer metastasis. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/14/3793/F1.large.jpg. Cancer Res; 78(14); 3793–808. ©2018 AACR.

Temporal data on gene expression and context-specific open chromatin states can improve identification of key transcription factors (TFs) and the gene regulatory networks (GRNs) controlling cellular differentiation. However, their integration remains challenging. Here, we delineate a general approach for data-driven and unbiased identification of key TFs and dynamic GRNs, called EPIC-DREM. We generated time-series transcriptomic and epigenomic profiles during differentiation of mouse multipotent bone marrow stromal cells (MSCs) towards adipocytes and osteoblasts. Using our novel approach we constructed time-resolved GRNs for both lineages. To prioritize the identified shared regulators, we mapped dynamic super-enhancers in both lineages and associated them to target genes with correlated expression profiles. We identified aryl hydrocarbon receptor (AHR) and Glis family zinc finger 1 (GLIS1) as mesenchymal key TFs controlled by dynamic MSC-specific super-enhancers that become repressed in both lineages. AHR and GLIS1 control differentiation-induced genes and we propose they function as guardians of mesenchymal multipotency.

We use 2nd generation sequencing technology on sedimentary ancient DNA (sedaDNA) from a lake in South Greenland to reconstruct the local floristic history around a low-arctic lake and compare the results with those previously obtained from pollen and macrofossils in the same lake. Thirty-eight of thirty-nine samples from the core yielded putative DNA sequences. Using a multiple assignment strategy on the trnL g–h DNA barcode, consisting of two different phylogenetic and one sequence similarity assignment approaches, thirteen families of plants were identified, of which two (Scrophulariaceae and Asparagaceae) are absent from the pollen and macrofossil records. An age model for the sediment based on twelve radiocarbon dates establishes a chronology and shows that the lake record dates back to 10,650 cal yr BP. Our results suggest that sedaDNA analysis from lake sediments, although taxonomically less detailed than pollen and macrofossil analyses can be a complementary tool for establishing the composition of both terrestrial and aquatic local plant communities and a method for identifying additional taxa.

We used high-sensitivity, high-resolution tandem mass spectrometry to shotgun sequence ancient protein remains extracted from a 43 000 year old woolly mammoth ( Mammuthus primigenius ) bone preserved in the Siberian permafrost. For the first time, 126 unique protein accessions, mostly low-abundance extracellular matrix and plasma proteins, were confidently identified by solid molecular evidence. Among the best characterized was the carrier protein serum albumin, presenting two single amino acid substitutions compared to extant African ( Loxodonta africana ) and Indian ( Elephas maximus ) elephants. Strong evidence was observed of amino acid modifications due to post-mortem hydrolytic and oxidative damage. A consistent subset of this permafrost bone proteome was also identified in more recent Columbian mammoth ( Mammuthus columbi ) samples from temperate latitudes, extending the potential of the approach described beyond subpolar environments. Mass spectrometry-based ancient protein sequencing offers new perspectives for future molecular phylogenetic inference and physiological studies on samples not amenable to ancient DNA investigation. This approach therefore represents a further step into the ongoing integration of different high-throughput technologies for identification of ancient biomolecules, unleashing the field of paleoproteomics.

Second-generation sequencing platforms have revolutionized the field of ancient DNA, opening access to complete genomes of past individuals and extinct species. However, these platforms are dependent on library construction and amplification steps that may result in sequences that do not reflect the original DNA template composition. This is particularly true for ancient DNA, where templates have undergone extensive damage post-mortem. Here, we report the results of the first “true single molecule sequencing” of ancient DNA. We generated 115.9 Mb and 76.9 Mb of DNA sequences from a permafrost-preserved Pleistocene horse bone using the Helicos HeliScope and Illumina GAIIx platforms, respectively. We find that the percentage of endogenous DNA sequences derived from the horse is higher among the Helicos data than Illumina data. This result indicates that the molecular biology tools used to generate sequencing libraries of ancient DNA molecules, as required for second-generation sequencing, introduce biases into the data that reduce the efficiency of the sequencing process and limit our ability to fully explore the molecular complexity of ancient DNA extracts. We demonstrate that simple modifications to the standard Helicos DNA template preparation protocol further increase the proportion of horse DNA for this sample by threefold. Comparison of Helicos-specific biases and sequence errors in modern DNA with those in ancient DNA also reveals extensive cytosine deamination damage at the 3′ ends of ancient templates, indicating the presence of 3′-sequence overhangs. Our results suggest that paleogenomes could be sequenced in an unprecedented manner by combining current second- and third-generation sequencing approaches.

International audience; The drug discovery process is a starving machine requiring constant feeding with new chemical compounds. Synthetic or natural scaffolds: what are the best sources? While synthetic molecules are rapidly generated by combinatorial chemistry, they show lower chemical diversity than their natural counterparts. A significant fraction of known natural products is issued from microbial secondary metabolism; however, more than 95% of bacterial organisms remain unexploited as a source of active chemical compounds due to their cultivation difficulties. Recent technological advances in metagenomics have provided reliable access to chemicals of these hidden bugs, thus opening up new opportunities for feeding the machine.

The current existing enzymes have been identified from cultivable micro-organisms, most frequently from bacteria. These bacterial biocatalytic capabilities have been widely used for biotransformations, resulting in the development of profitable industrial bioprocesses in the fields of feed and food processing, textiles, agro-chemistry, cosmetics, pharmaceuticals and fine chemistry. However, the originality of this bioresource is progressively drying up, while requests from industry for novel biocatalytic activities are increasing in the face of economic and environmental pressure. Metagenomics, through access to the huge reservoir of uncultivated bacteria which represents the majority of the present biodiversity, opens the door to new industrial sources of enzymes. Surmounting hurdles encountered with this technology (e.g. DNA extraction to obtain high quality DNA libraries with proper statistical representativity, setting up of relevant high throughput screenings assays, combining functional and genome-b...

Sarkissian, Clio Der et al., Przewalski’s horses (PHs, Equus ferus ssp. przewalskii) were discovered in the Asian steppes in the 1870s and represent the last remaining true wild horses. PHs became extinct in the wild in the 1960s but survived in captivity, thanks to major conservation efforts. The current population is still endangered, with just 2,109 individuals, one-quarter of which are in Chinese and Mongolian reintroduction reserves [1]. These horses descend from a founding population of 12 wild-caught PHs and possibly up to four domesticated individuals [2–4]. With a stocky build, an erect mane, and stripped and short legs, they are phenotypically and behaviorally distinct from domesticated horses (DHs, Equus caballus). Here, we sequenced the complete genomes of 11 PHs, representing all founding lineages, and five historical specimens dated to 1878–1929 CE, including the Holotype. These were compared to the hitherto-most-extensive genome dataset characterized for horses, comprising 21 new genomes. We found that loci showing the most genetic differentiation with DHs were enriched in genes involved in metabolism, cardiac disorders, muscle contraction, reproduction, behavior, and signaling pathways. We also show that DH and PH populations split ∼45,000 years ago and have remained connected by gene-flow thereafter. Finally, we monitor the genomic impact of ∼110 years of captivity, revealing reduced heterozygosity, increased inbreeding, and variable introgression of domestic alleles, ranging from non-detectable to as much as 31.1%. This, together with the identification of ancestry informative markers and corrections to the International Studbook, establishes a framework for evaluating the persistence of genetic variation in future reintroduced populations., This work was supported by the Danish Council for Independent Research, Natural Sciences (FNU-4002-00152B); the Danish National Research Foundation (DNFR94); the Villum Fonden Blokstipendium (2014); the Lundbeck Foundation (R52-A5062); the Israel Science Foundation (1365/10); the German Research Council (DFG-LU852/7-4); the NIH (R01-GM40282); the Caesar Kleberg Foundation for Wildlife Conservation; the John and Beverly Stauffer Foundation; FP7 European Marie-Curie programs (CIG-293845, ITN-290344, IEF-328024, IEF-299176, and IEF-302617); a National Science Foundation Graduate Research Fellowship; and the Human Frontier Science Program (LT000320/2014).

Summary: Ancient DNA extracts consist of a mixture of contaminant DNA molecules, most often originating from environmental microbes, and endogenous fragments exhibiting substantial levels of DNA damage. The latter introduce specific nucleotide misincorporations and DNA fragmentation signatures in sequencing reads that could be advantageously used to argue for sequence validity. mapDamage is a Perl script that computes nucleotide misincorporation and fragmentation patterns using next-generation sequencing reads mapped against a reference genome. The Perl script outputs are further automatically processed in embedded R script in order to detect typical patterns of genuine ancient DNA sequences. Availability and implementation: The Perl script mapDamage is freely available with documentation and example files at http://geogenetics.ku.dk/all_literature/mapdamage/. The script requires prior installation of the SAMtools suite and R environment and has been validated on both GNU/Linux and MacOSX operating systems. Contact: aginolhac@snm.ku.dk Supplementary information: Supplementary data available at Bioinformatics online

Schubert, Michael et al., The domestication of the horse ∼5.5 kya and the emergence of mounted riding, chariotry, and cavalry dramatically transformed human civilization. However, the genetics underlying horse domestication are difficult to reconstruct, given the near extinction of wild horses. We therefore sequenced two ancient horse genomes from Taymyr, Russia (at 7.4- and 24.3-fold coverage), both predating the earliest archeological evidence of domestication. We compared these genomes with genomes of domesticated horses and the wild Przewalski's horse and found genetic structure within Eurasia in the Late Pleistocene, with the ancient population contributing significantly to the genetic variation of domesticated breeds. We furthermore identified a conservative set of 125 potential domestication targets using four complementary scans for genes that have undergone positive selection. One group of genes is involved in muscular and limb development, articular junctions, and the cardiac system, and may represent physiological adaptations to human utilization. A second group consists of genes with cognitive functions, including social behavior, learning capabilities, fear response, and agreeableness, which may have been key for taming horses. We also found that domestication is associated with inbreeding and an excess of deleterious mutations. This genetic load is in line with the "cost of domestication" hypothesis also reported for rice, tomatoes, and dogs, and it is generally attributed to the relaxation of purifying selection resulting from the strong demographic bottlenecks accompanying domestication. Our work demonstrates the power of ancient genomes to reconstruct the complex genetic changes that transformed wild animals into their domesticated forms, and the population context in which this process took place. © 2014, National Academy of Sciences. All rights reserved., This work was supported by the Danish Council for Independent Research, Natural Sciences (FNU); the Danish National Research Foundation (DNFR94); a Marie-Curie Career Integration Grant (FP7 CIG-293845); and the International Research Group Program (IRG14-08), Deanship of Scientific Research (King Saud University, Saudi Arabia). H.J. was supported by a Marie-Curie Initial Training Network Grant (EUROTAST; FP7 ITN-290344); A.G. and L. Ermini were supported by Marie-Curie Intra-European Fellowships (FP7 IEF-299176 and FP7 IEF-302617); M.S. was supported by a Lundbeck Foundation Grant (R52-A5062); I.D., A.F., and L. Excoffier were supported by a Swiss National Science Foundation Grant (31003A-143393); M.H. was supported by a European Research Council (ERC) Consolidator Grant (310763); T.M.-B. was supported by an ERC Starting Grant (260372) and by a Ministerio de Ciencia e Innovación (MICINN) Grant (BFU2011-28549); B.S. was supported by the Packard Foundation; and D.C. was supported by start-up funds to B.S. from the University of California, Santa Cruz.

Horses, asses, and zebras belong to a single genus, Equus, which emerged 4.0–4.5 Mya. Although the equine fossil record represents a textbook example of evolution, the succession of events that gave rise to the diversity of species existing today remains unclear. Here we present six genomes from each living species of asses and zebras. This completes the set of genomes available for all extant species in the genus, which was hitherto represented only by the horse and the domestic donkey. In addition, we used a museum specimen to characterize the genome of the quagga zebra, which was driven to extinction in the early 1900s. We scan the genomes for lineage-specific adaptations and identify 48 genes that have evolved under positive selection and are involved in olfaction, immune response, development, locomotion, and behavior. Our extensive genome dataset reveals a highly dynamic demographic history with synchronous expansions and collapses on different continents during the last 400 ky after major climatic events. We show that the earliest speciation occurred with gene flow in Northern America, and that the ancestor of present-day asses and zebras dispersed into the Old World 2.1–3.4 Mya. Strikingly, we also find evidence for gene flow involving three contemporary equine species despite chromosomal numbers varying from 16 pairs to 31 pairs. These findings challenge the claim that the accumulation of chromosomal rearrangements drive complete reproductive isolation, and promote equids as a fundamental model for understanding the interplay between chromosomal structure, gene flow, and, ultimately, speciation.

Next-generation sequencing technologies have revolutionized the field of paleogenomics, allowing the reconstruction of complete ancient genomes and their comparison with modern references. However, this requires the processing of vast amounts of data and involves a large number of steps that use a variety of computational tools. Here we present PALEOMIX (http://geogenetics.ku.dk/publications/paleomix), a flexible and user-friendly pipeline applicable to both modern and ancient genomes, which largely automates the in silico analyses behind whole-genome resequencing. Starting with next-generation sequencing reads, PALEOMIX carries out adapter removal, mapping against reference genomes, PCR duplicate removal, characterization of and compensation for postmortem damage, SNP calling and maximum-likelihood phylogenomic inference, and it profiles the metagenomic contents of the samples. As such, PALEOMIX allows for a series of potential applications in paleogenomics, comparative genomics and metagenomics. Applying the PALEOMIX pipeline to the three ancient and seven modern Phytophthora infestans genomes as described here takes 5 d using a 16-core server.

Motivation: Ancient DNA (aDNA) molecules in fossilized bones and teeth, coprolites, sediments, mummified specimens and museum collections represent fantastic sources of information for evolutionary biologists, revealing the agents of past epidemics and the dynamics of past populations. However, the analysis of aDNA generally faces two major issues. Firstly, sequences consist of a mixture of endogenous and various exogenous backgrounds, mostly microbial. Secondly, high nucleotide misincorporation rates can be observed as a result of severe post-mortem DNA damage. Such misincorporation patterns are instrumental to authenticate ancient sequences versus modern contaminants. We recently developed the user-friendly mapDamage package that identifies such patterns from next-generation sequencing (NGS) sequence datasets. The absence of formal statistical modeling of the DNA damage process, however, precluded rigorous quantitative comparisons across samples. Results: Here, we describe mapDamage 2.0 that extends the original features of mapDamage by incorporating a statistical model of DNA damage. Assuming that damage events depend only on sequencing position and post-mortem deamination, our Bayesian statistical framework provides estimates of four key features of aDNA molecules: the average length of overhangs (λ), nick frequency (ν) and cytosine deamination rates in both double-stranded regions () and overhangs (). Our model enables rescaling base quality scores according to their probability of being damaged. mapDamage 2.0 handles NGS datasets with ease and is compatible with a wide range of DNA library protocols. Availability: mapDamage 2.0 is available at ginolhac.github.io/mapDamage/ as a Python package and documentation is maintained at the Centre for GeoGenetics Web site (geogenetics.ku.dk/publications/mapdamage2.0/). Contact: jonsson.hakon@gmail.com Supplementary information: Supplementary data are available at Bioinformatics online.

The genus Equus is richly represented in the fossil record, yet our understanding of taxonomic relationships within this genus remains limited. To estimate the phylogenetic relationships among modern horses, zebras, asses and donkeys, we generated the first data set including complete mitochondrial sequences from all seven extant lineages within the genus Equus. Bayesian and Maximum Likelihood phylogenetic inference confirms that zebras are monophyletic within the genus, and the Plains and Grevy’s zebras form a well-supported monophyletic group. Using ancient DNA techniques, we further characterize the complete mitochondrial genomes of three extinct equid lineages (the New World stilt-legged horses, NWSLH; the subgenus Sussemionus; and the Quagga, Equus quagga quagga). Comparisons with extant taxa confirm the NWSLH as being part of the caballines, and the Quagga and Plains zebras as being conspecific. However, the evolutionary relationships among the non-caballine lineages, including the now-extinct subgenus Sussemionus, remain unresolved, most likely due to extremely rapid radiation within this group. The closest living outgroups (rhinos and tapirs) were found to be too phylogenetically distant to calibrate reliable molecular clocks. Additional mitochondrial genome sequence data, including radiocarbon dated ancient equids, will be required before revisiting the exact timing of the lineage radiation leading up to modern equids, which for now were found to have possibly shared a common ancestor as far as up to 4 Million years ago (Mya).

BackgroundSecond-generation sequencing technologies have revolutionized our ability to recover genetic information from the past, allowing the characterization of the first complete genomes from past individuals and extinct species. Recently, third generation Helicos sequencing platforms, which perform true Single-Molecule DNA Sequencing (tSMS), have shown great potential for sequencing DNA molecules from Pleistocene fossils. Here, we aim at improving even further the performance of tSMS for ancient DNA by testing two novel tSMS template preparation methods for Pleistocene bone fossils, namely oligonucleotide spiking and treatment with DNA phosphatase.ResultsWe found that a significantly larger fraction of the horse genome could be covered following oligonucleotide spiking however not reproducibly and at the cost of extra post-sequencing filtering procedures and skewed %GC content. In contrast, we showed that treating ancient DNA extracts with DNA phosphatase improved the amount of endogenous sequence information recovered per sequencing channel by up to 3.3-fold, while still providing molecular signatures of endogenous ancient DNA damage, including cytosine deamination and fragmentation by depurination. Additionally, we confirmed the existence of molecular preservation niches in large bone crystals from which DNA could be preferentially extracted.ConclusionsWe propose DNA phosphatase treatment as a mechanism to increase sequence coverage of ancient genomes when using Helicos tSMS as a sequencing platform. Together with mild denaturation temperatures that favor access to endogenous ancient templates over modern DNA contaminants, this simple preparation procedure can improve overall Helicos tSMS performance when damaged DNA templates are targeted.

National audience

International audience; Abstract. Type I polyketide synthases (PKSI) are modular multidomain enzymes involved in the biosynthesis of many natural products of industrial interest. PKSI modules are minimally organized in three domains: ketosynthase (KS), acyltransferase (AT), and acyl carrier protein. The KS domain phylogeny of 23 PKSI clusters was determined. The results obtained suggest that many horizontal transfers of PKSI genes have occurred between actinomycetales species. Such gene transfers may explain the homogeneity and the robustness of the actinomycetales group since gene transfers between closely related species could mimic patterns generated by vertical inheritance. We suggest that the linearity and instability of actinomycetales chromosomes associated with their large quantity of genetic mobile elements have favored such horizontal gene transfers.

The metagenomic approach provides direct access to diverse unexplored genomes, especially from uncultivated bacteria in a given environment. This diversity can conceal many new biosynthetic pathways. Type I polyketide synthases (PKSI) are modular enzymes involved in the biosynthesis of many natural products of industrial interest. Among the PKSI domains, the ketosynthase domain (KS) was used to screen a large soil metagenomic library containing more than 100,000 clones to detect those containing PKS genes. Over 60,000 clones were screened, and 139 clones containing KS domains were detected. A 700-bp fragment of the KS domain was sequenced for 40 of 139 randomly chosen clones. None of the 40 protein sequences were identical to those found in public databases, and nucleic sequences were not redundant. Phylogenetic analyses were performed on the protein sequences of three metagenomic clones to select the clones which one can predict to produce new compounds. Two PKS-positive clones do not belong to any of the 23 published PKSI included in the analysis, encouraging further analyses on these two clones identified by the selection process.

Sialic acid-binding receptors (Siglecs) are linked to neurodegenerative processes, but the role of sialic acids in physiological aging is still not fully understood. We investigated the impact of reduced sialylation in the brain of mice heterozygous for the enzyme glucosamine-2-epimerase/N-acetylmannosamine kinase (GNE+/-) that is essential for sialic acid biosynthesis. We demonstrate that GNE+/- mice have hyposialylation in different brain regions, less synapses in the hippocampus and reduced microglial arborization already at 6 months followed by increased loss of neurons at 12 months. A transcriptomic analysis revealed no pro-inflammatory changes indicating an innate homeostatic immune process leading to the removal of synapses and neurons in GNE+/- mice during aging. Crossbreeding with complement C3-deficient mice rescued the earlier onset of neuronal and synaptic loss as well as the changes in microglial arborization. Thus, sialic acids of the glycocalyx contribute to brain homeostasis and act as a recognition system for the innate immune system in the brain.

Background Next-Generation Sequencing has revolutionized our approach to ancient DNA (aDNA) research, by providing complete genomic sequences of ancient individuals and extinct species. However, the recovery of genetic material from long-dead organisms is still complicated by a number of issues, including post-mortem DNA damage and high levels of environmental contamination. Together with error profiles specific to the type of sequencing platforms used, these specificities could limit our ability to map sequencing reads against modern reference genomes and therefore limit our ability to identify endogenous ancient reads, reducing the efficiency of shotgun sequencing aDNA. Results In this study, we compare different computational methods for improving the accuracy and sensitivity of aDNA sequence identification, based on shotgun sequencing reads recovered from Pleistocene horse extracts using Illumina GAIIx and Helicos Heliscope platforms. We show that the performance of the Burrows Wheeler Aligner (BWA), that has been developed for mapping of undamaged sequencing reads using platforms with low rates of indel-types of sequencing errors, can be employed at acceptable run-times by modifying default parameters in a platform-specific manner. We also examine if trimming likely damaged positions at read ends can increase the recovery of genuine aDNA fragments and if accurate identification of human contamination can be achieved using a strategy previously suggested based on best hit filtering. We show that combining our different mapping and filtering approaches can increase the number of high-quality endogenous hits recovered by up to 33%. Conclusions We have shown that Illumina and Helicos sequences recovered from aDNA extracts could not be aligned to modern reference genomes with the same efficiency unless mapping parameters are optimized for the specific types of errors generated by these platforms and by post-mortem DNA damage. Our findings have important implications for future aDNA research, as we define mapping guidelines that improve our ability to identify genuine aDNA sequences, which in turn could improve the genotyping accuracy of ancient specimens. Our framework provides a significant improvement to the standard procedures used for characterizing ancient genomes, which is challenged by contamination and often low amounts of DNA material.