Supplemental Digital Content is available in the text The DEFINITION (Impact of the complexity of bifurcation lesions treated with drug-eluting stents) study has provided a novel classification to evaluate the complexity of coronary bifurcation lesion according to coronary angiography, but angiographic imaging due to its low resolution and inherited limitation may result in an inaccurate adjudication.We used optical coherence tomography (OCT) to further evaluate the coronary characteristics in a patient with “simple” bifurcation lesion which was classified by the DEFINITION criteria. However, a “complex” bifurcation lesion was defined and confirmed according to the OCT results.A double kissing Crush stenting approach was adopted to treat this “complex” case finally. The immediate and long-term angiographic and OCT results were excellent.OCT may be useful imaging modality to classify complexity of coronary bifurcation lesion and subsequently guide its treatment strategy. The DEFINITION (Impact of the complexity of bifurcation lesions treated with drug-eluting stents) study has provided evidence that complex bifurcation stenting—and in particular double kissing (DK) Crush technique—is associated with better clinical outcomes in lesions with a specific anatomical characteristics assessed by coronary angiography. However, coronary angiography may often fail to evaluate coronary artery pathology. In this case report, we used for the first time optical coherence tomography (OCT)—rather than coronary angiography—to classify lesion severity according to the DEFINITION criteria and assess the immediate and long term results of a DK Crush stenting.The treatment of coronary bifurcation lesions can be challenging and associated with a high incidence of major adverse cardiac events. The Consensus of the European Bifurcation Club recommends provisional stenting as the preferred technique for the majority of bifurcation lesions.2A 50-year-old male patient with a history of hypertension, current smoker, and hyperlipidemia was admitted with angina symptoms that appeared 2 weeks ago. There was no positive finding from the relevant physical examination. Electrocardiogram demonstrated ST-segment elevations in leads V1 to V4, and the troponin was raised. The patient diagnosed with an acute myocardial infarction was treated conservatively in the local hospital and transferred to our center for coronary angiography/percutaneous coronary intervention (PCI).Coronary angiography showed a thrombotic occlusion in the proximal left anterior descending (LAD) artery Fig. . The lesA 2.75 × 33 mm drug-eluting stent was implanted in the mid-LAD artery before a DK Crush stenting approach adopted to treat this “complex” case see Fig. , D1. TheAt 1-year follow-up, no major adverse cardiac events were reported. Angiographic examination showed no obvious lumen obstruction within the stented segment. OCT and offline 3-dimensional reconstruction demonstrated that stent struts in both LAD and D2 were well covered by a thin neointimal layer that did not cause significant obstruction  which presented stent strut malapposition, major dissection, major tissue prolapsed, or intracoronary thrombus. Nevertheless, there are limited data about the clinical value of OCT in guiding PCI. Although recent registries have shown potential benefit of OCT guidance, there are no data in the context of randomized controls trials that would support its use in the clinical setting. Futures studies including the ILLUMINA III (NCT 2471586) and OPINION (NCT 01873027) are anticipated to provide further evidence about the clinical value of the OCT guiding PCI.In the past years, OCT imaging contributed tremendously to the optimization of bifurcation stenting techniques. With the high resolution, OCT could facilitate interventionlists to re-cross proper stent cell, which is the key procedure in both provisional stenting and 2-stent techniques.


Rivularia, which dominate in the mat. It occurred via organo-mineralization processes within a large EPS matrix excreted by the diverse microbial consortium associated with Rivularia where diatoms and anoxygenic phototrophic bacteria were particularly abundant. By structuring a large microbial consortium, Rivularia should then favor the formation of organic-rich laminations of carbonates that can be preserved in stromatolites. By using Fourier Transform Infrared spectroscopy and Synchrotron-based deep UV fluorescence imaging, we compared laminations rich in structures resembling Rivularia to putatively chemically-precipitated laminations in oncoids associated with the mats. We showed that they presented a different mineralogy jointly with a higher content in organic remnants, hence providing some criteria of biogenicity to be searched for in the fossil record.Stromatolites are organo-sedimentary structures that represent some of the oldest records of the early biosphere on Earth. Cyanobacteria are considered as a main component of the microbial mats that are supposed to produce stromatolite-like structures. Understanding the role of cyanobacteria and associated microorganisms on the mineralization processes is critical to better understand what can be preserved in the laminated structure of stromatolites. Laguna Negra , a high-altitude hypersaline lake where stromatolites are currently formed, is considered as an analog environment of early Earth. This study aimed at characterizing carbonate precipitation within microbial mats and associated oncoids in Laguna Negra. In particular, we focused on carbonated black pustular mats. By combining Confocal Laser Scanning Microscopy, Scanning Electron Microscopy, Laser Microdissection and Whole Genome Amplification, Cloning and Sanger sequencing, and Focused Ion Beam milling for Transmission Electron Microscopy, we showed that carbonate precipitation did not directly initiate on the sheaths of cyanobacterial  Microbialites record microbial activities and sedimentary processes under a lithified form  restrict eukaryotic life. Lithification is controlled by environmental processes , powder X-Ray Diffraction (XRD) and Synchrotron-based deep UV fluorescence imaging (S-DUV), we clearly highlighted that these laminations present a different pattern compared to putatively chemically-formed laminations, hence providing for laminations some criteria of biogenicity to be searched for in the fossil record.The main objectives of this study were (i) to investigate at Laguna Negra the potential role of a microbial consortium forming the black pustular mat (BP) on the initiation of carbonate precipitation and lamination formation and (ii) to identify biogenicity criteria for the corresponding laminations in subfossil oncoids. By jointly using Confocal Laser Scanning Microscopy (CLSM), Scanning and Transmission Electron Microscopy (SEM/TEM) associated with Energy Dispersive X-ray Spectrometry (EDXS) and Focused Ion Beam (FIB) milling, as well as phylogenetic analyses on the bulk BP mat and on laser microdissected mat cells, we highlighted the presence of an interesting microbial consortium associated with 2 is observed. This belt mostly consists of oncoids and associated microbial mats with morphological and textural differences described in Gomez et al. . Samples for SEM-EDXS were fixed in the laboratory with a 2% glutaraldehyde solution and stored in the dark at 4°C for 2 h. After fixation, samples were washed and progressively dehydrated in a gradual series of ethanol and water baths at increasing ethanol concentrations , prior to air drying or critical point drying . Samples for CLSM were fixed in 4% paraformaldehyde solution directly in the field at 4°C, then washed in phosphate buffered saline (PBS) once back in the laboratory and stored in (1/1) ethanol/PBS solution at −20°C until use. Oncoids and microbialites were also collected and stored at 4°C without any chemical fixation.The Laguna Negra is a high altitude lake  located in the Puna region of the Catamarca Province, Argentina Figure . At the z et al.  -aminomethyl]-fluorescein) produces a stable fluorescent complex in the presence of calcium, strontium, barium, and magnesium ions , a green fluorescent nucleic acid dye. Syto®9-stained samples and resin-embedded microbial mat samples were examined at the Institut de Physique du Globe de Paris  using a FluoView™ FV1000 confocal laser scanning microscope with a spectral resolution of 2 nm and a spatial resolution of 0.2 μm (Olympus). The microscope was equipped with a 405 nm laser diode, and multi-line argon , helium-neon-green (543 nm) and helium-neon-red (633 nm) lasers. Fluorescence images were obtained with concomitant excitation at wavelengths of 405, 488, and 543 nm by collecting the emitted fluorescence between 425 and 475 nm, 500 and 530 nm, and 560 and 660 nm, respectively. For CSLM image acquisitions on resin-embedded sections, a water immersion LUMPLFL 60XW objective  with a numerical aperture (N.A.) of 0.9 was used. For fresh biofilms examination, an oil immersion objective UPLSAPO 60XO  was used. 3D images were acquired, visualized, and processed using the F10-ASW FLUOVIEW software (Olympus).MH084957 to MH084967 for cyanobacteria, from MH109330 to 109371 for bacteria, and from MH127632 to MH127645 for archaea.DNA extractions were performed using the MoBio PowerSoil® DNA kit (MoBio) following the instructions provided by the manufacturer. Bacterial 16S rRNA genes were amplified by polymerase chain reaction (PCR) using the bacterial specific primer 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) combined with the universal prokaryotic reverse primer 1492R (5′-GGTTACCTTGTTACGACTT-3′). Archaeal 16S rRNA genes were amplified using the archaeal specific primer 21F (5′-TTCCGGTTGATCCTGCCGGA-3′) and the prokaryote specific reverse primer 1492R. Cyanobacteria 16S rRNA genes were amplified using the cyanobacterial specific primers Cya-106F (5′-CGGACGGGTGAGTAACGCGTGA-3′) and the cyanobacterial specific reverse primer Cya-1387R (5′-TAACGACTTCGGGCGTGACC-3′). One microliter of the extracted DNA was used in a reaction buffer volume of 25 μL containing dNTPs (10 nmol each), 20 pmol of each primer and 1 U GoTaq polymerase (Promega). PCR reactions were performed under the following conditions: 35 cycles  preceded by 2 min denaturation at 94°C, and followed by 7 min extension at 72°C. Cloning was done using the Topo® TA Cloning® system (ThermoFisher Scientific) following the instructions provided by the manufacturer. After plating, positive clones were screened by PCR amplification of inserts using flanking vector primers and the PCR products were partially sequenced (≈700 bp) by GATC Biotech using flanking vector primer T7 (5′-TAATACGACTCACTATAGGG-3′). At least one representative clone per phylotype or Operational Taxonomic Unit  was fully sequenced for Cyanobacteria using flanking vector primer M13R (5′-CAGGAAACAGCTATGAC-3′) for detailed phylogenetic analysis. The sequences have been deposited at the GenBank database and correspond to the accession numbers MH119800 to MH119817.Filamentous cyanobacterial cells were isolated using a Zeiss PALM MicroBeam apparatus (Carl Zeiss NTS GmbH) installed in a sterile room at IPGP. We then used the REPLI-g Single Cell Kit (Qiagen) to amplify whole genomic DNA of the microdissected cells. Bacterial 16S rRNA encoding genes were then amplified by PCR using the bacterial specific primer 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) combined with the prokaryote specific reverse primer 1492R (5′-GGTTACCTTGTTACGACTT-3′). One microliter of 1/10 diluted amplified genomic DNA was used in a reaction buffer volume of 25 μL containing dNTPs (10 nmol each), 20 pmol of each primer and 1U of GoTaq polymerase (Promega). PCR reactions were performed under the following conditions: 35 cycles  preceded by 2 min denaturation at 94°C, and followed by 7 min extension at 72°C. Cloning and Sanger sequencing were done as previously described for the 16S rRNA encoding genes retrieved from the bulk DNA extraction. The corresponding sequences have been deposited at the GenBank database and accession numbers range from pufL and pufM genes coding for subunits of the photosynthetic reaction center of aerobic anoxygenic photosynthetic bacteria  and the pufLM-R primer (5′-CCCATSGTCCAGCGCCAGAA-3′)  following the instructions provided by the manufacturer and the PCR products were totally sequenced by GATC Biotech using flanking vector primer T7 (5′-TAATACGACTCACTATAGGG-3′) and M13R (5′-CAGGAAACAGCTATGAC-3′). The corresponding sequences have been deposited in the GenBank database and accession numbers range from MH101761 to MH101777.Partial Taxonomic affiliations were obtained using BLAST  at the X Ray Analysis Laboratory . SEM observation on carbon-coated samples dried at the critical point were also performed at the Service Commun de Microscopie Electronique à Balayage  using a Zeiss Supra 55VP (Carl Zeiss NTS GmbH) SEM equipped with an EDXS spectrometer . Images were collected using secondary electron detectors  and a backscattered electron detector (AsB). Accelerating voltage ranged from 3 to 15 kV at variable pressures and high current (up to 1 nA).Powder X-ray diffraction (XRD) analyses were conducted on BP microbial mat and lithified BP samples using a Philips PW1800/10 powder diffractometer equipped with a Cu anode and a graphite monochromator (LAMARX). Measurements were performed at 40 kV voltage and 30 mA current, from 10 to 60° (2 θ), with an acquisition time of 2.0 s and an angular step of 0.100° 2θ. Resulting diffractograms were analyzed using Highscore software.+ beam operated at 20 nA was used for the initial steps of the milling. Progressive excavation from both sides of the section area was performed through repeated milling steps. Depth of milling was approximately 7 microns. The final thinning of the section was performed with a less intense Ga+ beam operated at 100 pA current.Eight ultrathin electron-transparent sections (~100 nm in thickness) were prepared by FIB milling on an Auriga® FIB-SEM (Carl Zeiss NTS GmbH) available at IPGP using the FIB “lift out” technique  observations were carried out on all FIB sections using a JEOL 2100F microscope (JEOL Ltd.) operating at 200 kV at the Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie . The TEM is equipped with a field emission gun, a ultrahigh resolution UHR pole piece and a Gatan energy filter. High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) was used with a focused electron beam of a few nm for Z-contrast imaging. Energy Dispersive X-ray Spectrometry (EDXS) analyses were performed using a JEOL detector equipped with an ultrathin window that allowed detection of low Z elements.−1 range, with a spot size of 20 × 20 μm, a spectral resolution of 8 cm−1 and with 64 accumulations per spectrum. Background spectra were acquired between each spectrum under the same analytical conditions. Data were processed using the OMNIC™ software (Thermo Fisher Scientific).Cross-sections of oncoids associated with the black pustular mats were obtained using a sterilized diamond saw and sterile deionized water. They were prepared as doubly-polished sections of about 100 μm thick and without any resin or glue in order to avoid organic contamination. FTIR hyperspectral images were acquired at IPGP using a Thermo Scientific iN10 MX microscope  with a liquid nitrogen cooled detector. Maps were collected in Attenuated Total Reflection (ATR) mode with a germanium tip. Punctual analyses were performed in the 4,000–700 cmRivularia-rich laminations in order to better describe the nature and spatial relationships between mineral phases and organic remnants at the micrometer scale. Both TELEMOS and POLYPHEME end stations available on the beamline were used. Samples were first investigated by full-field luminescence microscopy (TELEMOS end station) using an Axio ObserverZ1 microscope (Carl Zeiss MicroImaging) with a × 40 objective. The excitation wavelength was set at 275 nm with a DM300 dichroic mirror. Fluorescence emission was collected using 3 filters . Acquisition time was set at 30 s for all channels except for the 327–353 nm filter (60 s) and 2D fluorescence images were acquired along a transect, perpendicularly to the laminations of the oncoid sample. Images obtained with TELEMOS microscope were treated with ImageJ software  on an Olympus IX71 inverted microscope with homemade replacement of the intermediate lenses set to be transparent in the deep UV range  and full-field imaging at the French national synchrotron radiation facility SOLEIL  on the DISCO beamline  and measuring around 12 to 15 μm in diameter.The microbial mat was characterized by a bulbous to pustular morphology Figure  and measRivularia filaments (15–20 μm in diameter) were observed in petrographic sections of associated oncoids  and filamentous cells were also observed, fluorescing in green after Syto®9 staining. Among them, colonies of cells with a peculiar coccus shape (c1) were detected in close association with the Rivularia sheaths and the minerals  and Rivularia atra BIR MGR1 (AM230675), a marine species forming black colonies . 14 sequences represented by 3c-38 share 100% identities with Rivularia sp. PUNA_NP3_PCI185B (KY296608) isolated from Laguna Negra microbial mat. Heterotrophic Bacteroidetes affiliated to Marivirga  of cyanobacteria affiliated to the Rivularia genus were detected in both mats collected in 2013 and 2015 using cyanobacterial specific primers  were identical to the 3c-25 sequences detected after whole genome amplification . 17 sequences from 2015 were also closely related to sequences detected in microbialites from Alchichica, an alkaline Mexican lake .After bulk DNA extraction, cyanobacteria affiliated to the s Figure  but not n Figure . Yet, th5 Figure . The cloLewinella genus were identified. Their cultivable representatives  correspond to orange to black chemoorganotrophic bacteria . Finally, Verrucomicrobia members affiliated to the Haloferula genus were distinguished. They were close to Haloferula chungangensis (NR_109435), a heterotrophic ureolytic bacterium from marine sediments , which correspond to dark orange bacteria isolated from marine solar saltern , the Octadecabacter genus (98% identity with KX073749) and the Roseinatronobacter genus (97% identities with KJ486297), all these bacteria being aerobic phototrophic bacteria.In the BP mat collected in 2015, several detected bacteria were closely related to uncultivated bacteria from the Guerrero Negro hypersaline microbial mats belonging to Bacteroidetes (JN453999), Alphaproteobacteria  and Verrucomicrobia (JN480742). Other identified Bacteroidetes were close to bacteria from El Tatio hot springs (GU437622), a high altitude (4400 meters above sea level) geothermal site with low sulfide and high arsenic concentrations in Chile , a strain using H2 and formate for growth and methane production  a marine methanogenic archaea , which uses hydrogen and carbon dioxide as substrates for growth  showed that at least 18 different sequences of aerobic anoxygenic phototrophic bacteria (AAnPB) were present in the analyzed BP mat. As also noticed for the 16S rRNA encoding genes, two groups of AAnPB seemed to be endemic of high altitude Andean lakes as the partial pufLM sequences were close to sequences detected in a high altitude salt lake in Chile (FN813741 and FN813748)  Figure .Rivularia filaments were observed in the superficial pustular zone of the BP mats. They showed a tangled arrangement with variable amounts of EPS-associated mineral aggregates and diatoms located between the cyanobacterial filaments  already observed in Figure Rivularia sheaths  presented a similar pattern and are summarized in Figure 32− vibrational bands  were compared to carbonate standards and mainly corresponded to calcite , along with a marked shoulder in the ν3 asymmetric stretching band  and/or a different mineralogy.The mineralogy of t Figure  and indit Figure . Most ofRivularia filaments in the carbonate precipitation process. Thus, the role of Rivularia and of the related consortium in the mineralization processes is discussed. In parallel, the composition of the corresponding laminations in oncoids, comparatively to other laminations, is discussed in order to characterize potential biosignatures to be searched for in fossil stromatolites.The different microscopic observations and microbial diversity analyses carried out in this study highlight the likely influence of the microbial consortium observed around Phormidium and Rivularia genus. The large filamentous cyanobacteria of around 15 μm in diameter, observed by optical microscopy and CLSM, were identified as Rivularia using laser microdissection  or to the cell wall domains to control the flux of calcium in and out of cells.The most abundantly detected cyanobacterial 16S rRNA gene sequences using the cyanobacterial specific primer were affiliated to the on Table . A striks Figure . It suggRivularia cyanobacteria and contrary to what was described for freshwater Rivularia  (Figures Rivularia sheath. Diatoms and other microorganisms such as Myxococcales and methanogenic archaea are known to produce large amounts of EPS (Bapteste et al., Rivularia, diatoms may also have promoted local alkalinization. Furthermore, some of the bacteria identified in the whole microbial community may present potential alkalinizing metabolisms favoring carbonate precipitation. Among them are the Myxococcales identified in the BP mats of Laguna Negra. Some bacteria belonging to this order are known to favor mineral precipitation (Jimenez-López et al., Myxococcus xanthus, which induces carbonate precipitation by ammonification, resulting in an increase of the alkalinity in the culture medium (González-Muñoz et al., c1, Figure Roseobacter clade (Figure pufLM encoding genes showed that specific AAnPB develop in hypersaline high altitude Andean lakes. Members of the Roseobacter clade are known to interact with marine phytoplankton, including diatoms, also abundant in Andean lakes (Maidana and Seeligmann, Roseobacter-related species are able to use glycolate excreted by eukaryotic phytoplankton during autotrophic photorespiration (Fogg, Roseobacter clade are phototrophic bacteria but lack genes for inorganic carbon fixation (Lenk et al., via alkalinization linked to photoautotrophy (Dupraz and Visscher, Roseobacter clade are ureolysers and some are denitrifyers (Luo and Moran, Roseobacter denitrificans strain was increased by light stimulus (Doi and Shioi, On the contrary, the results obtained in this study indicated that carbonate precipitation occurred within the EPS matrix excreted by the diverse bacterial consortium associated with  Figures , 7 but ne Figure . Abundane Figure . The phyon Fogg, . MembersRivularia cyanobacteria favored the development of a large microbial consortium that indirectly promoted carbonate precipitation via organo-mineralization processes, and hence participated to the formation of stromatolites. The corresponding laminations in oncoids should then be considered as biologically-induced. They may present some direct or indirect markers of biological activity or cell remnants that may furnish biosignatures that could be searched for in the geological record. We then investigated stromatolites associated to the black pustular mat at Laguna Negra. We focused on laminations that presented Rivularia-like encrusted cells and we compared them to micritic and botryoidal laminations of the stromatolites, which may have a purely chemical origin. The laminations of interest presented well-preserved filaments that may have been encrusted in vivo or rapidly after death while preserving the original sheath morphology and sizes (Riding, Rivularia-rich laminations of oncoids associated with BP mats using S-DUV also clearly highlighted a different fluorescence signal compared to other laminations after excitation in the deep UV range (Figure Rivularia-like laminations seemed to indicate a possible higher proportion of aragonite relatively to calcite or Mg-calcite in these areas compared to other laminations. This could partly explain the different fluorescence pattern of this lamination. In that case, the presence of aragonite-rich laminations relatively to calcite-rich laminations may be a clue of biological activity (Lepot et al., −1 (Jamme et al., Rivularia sheaths, which favor the development of a large microbial consortium and are quite resistant to degradation, may have induced the preservation of a larger amount of biological organic remnants during mineralization comparatively to other laminations. It has important consequences for the search for biological traces in the fossil record. Indeed, these results are consistent with Lepot et al. (Rivularia-like structures presenting both higher aragonite together with a high organic content should then be tracked in order to decipher the biological origin of some laminations in fossil stromatolites.Overall,  Riding, . The mor Riding, , hence c Riding, . Pigment Riding, . They co Riding, . The anae Figure . The orit et al.  who assuRivularia sheath or in the Rivularia sheath-cell interspace. This could be respectively related to the activity of epiphytic bacteria and to the capacity of Rivularia to locally change calcium concentrations by shedding a possible mineralized S-layer or by maintaining a positive surface potential to avoid Ca2+ adsorption. However, Rivularia play a critical role in favoring and structuring the development of a large microbial consortium excreting a well-developed EPS matrix. Carbonate precipitation then occurred via organo-mineralization processes, the EPS matrix serving as a template for mineral nucleation. In addition to the alkalinizing activity of Rivularia and diatoms, the presence of putative AAnPB of the Roseobacter clade suggests that ureolysis and denitrification can be important metabolisms triggering carbonate precipitation by favoring local alkalinization. With progressive precipitation, Rivularia get entombed with intact sheath and photosynthetic pigments. The microspectroscopic analyses of the corresponding laminations rich in Rivularia-sheaths structures in stromatolites indicate that a close combination of aragonite and a high organic content should then be considered as potential evidence of microbially-mediated processes of formation of stromatolites, providing biosignatures to be searched for in the fossil record.By combining microscopic observations jointly with phylogenetic analyses, this study provides potential pathways for carbonate precipitation in the black pustular mat at Laguna Negra. Mineralization does not initiate directly on the EG, EM, and CP were responsible for the design of the study. EG, EM, CP, BM, LL, and FG performed experimental procedures and collected data. EG, KB, FG, and EM conducted FIB section, SEM and TEM. EG, EM, and LL performed DNA extraction and PCR. CP and EG performed FTIR analyses; CP, EG, BM, EM, FJ, and MR performed synchrotron-based Deep UV imaging experiment. ES performed XRD analyses. EM, CP, and EG interpreted the findings and drafted the manuscript. All authors critically reviewed content and approved the final version for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


P2RX3, which is associated with nociception, a peripheral pain response. We found ethanol significantly reduced the level of P2RX3 in undifferentiated hESCs, but induced the level of P2RX3 mRNA and protein in hESC-derived NPCs. Our result suggests ethanol-induced dysregulation of P2RX3 along with alterations in molecules involved in neural activity such as neuroactive ligand-receptor interaction may be a molecular event associated with alcohol-related peripheral neuropathy of an enhanced nociceptive response.Adverse effect of alcohol on neural function has been well documented. Especially, the teratogenic effect of alcohol on neurodevelopment during embryogenesis has been demonstrated in various models, which could be a pathologic basis for fetal alcohol spectrum disorders (FASDs). While the developmental defects from alcohol abuse during gestation have been described, the specific mechanisms by which alcohol mediates these injuries have yet to be determined. Recent studies have shown that alcohol has significant effect on molecular and cellular regulatory mechanisms in embryonic stem cell (ESC) differentiation including genes involved in neural development. To test our hypothesis that alcohol induces molecular alterations during neural differentiation we have derived neural precursor cells from pluripotent human ESCs in the presence or absence of ethanol treatment. Genome-wide transcriptomic profiling identified molecular alterations induced by ethanol exposure during neural differentiation of hESCs into neural rosettes and neural precursor cell populations. The Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis on significantly altered genes showed potential ethanol’s effect on JAK-STAT signaling pathway, neuroactive ligand-receptor interaction, Toll-like receptor (TLR) signaling pathway, cytokine-cytokine receptor interaction and regulation of autophagy. We have further quantitatively verified ethanol-induced alterations of selected candidate genes. Among verified genes we further examined the expression of  Alcohol consumption is recognized as the leading preventable cause of birth defects and mental retardation. High levels of alcohol consumption during pregnancy can result in fetal alcohol spectrum disorders (FASDs), which is characterized by prenatal and postnatal growth restriction, craniofacial dysmorphology and structural abnormalities of the central nervous system . DependiThe effect of alcohol on development has been widely studied in many different animal species . AdverseCurrently, it is not clearly established what causes FASDs. Recently, epigenetic regulations have emerged as potential mechanisms associated with alcohol teratogenesis. Epigenetic imprinting or genome-wide epigenetic reprogramming has been proposed as a mechanism responsible for alcohol-induced teratogenesis in preimplantation embryos , 3. Intein vivo molecular/cellular study models are not available.Embryonic stem cells (ESCs) are pluripotent cells that can be derived into all lineages of cells in the organism . Due to Stem cells are especially vulnerable to ethanol (EtOH) toxicity through decreases in pluripotency, survival capacity, and/or altered differentiation . Studiesin vitro neural stem cell model will be useful for the evaluation of developmental regulatory mechanisms and also the significance of their alterations in pathological conditions such as developmental disorders or exposure to neuroteratogens  diluted in 1X PBS (or blocking solution) and incubated overnight at 4°C. As a negative control, we have included the same isotype rabbit IgG or mouse IgG at equivalent concentrations. After washing three times for 5 min each with 1X PBS, samples were incubated with appropriate secondary antibody [donkey anti-rabbit IgG-FITC (1:500) or donkey anti-mouse IgG-rhodamine (1:500)] for 2 hrs at RT. After washing, the slips were mounted on the slide with mounting medium containing DAPI  and fluorescence imaging was performed with Olympus IX81 imaging microscope.RNA isolation and microarray analysis was done as described , 24. Bri2O by 1:4) and 1.5 μl of diluted cDNA was used per well (in 10 μl reaction volume) in a 384 well plate using LightCycler 480 SYBR Green I master mix .For validation of gene expression by quantitative RT-PCR analysis was performed as describe previously . Total RTo establish a model system to examine the molecular effects of EtOH on neural differentiation and maintenance of neural stem cells, we used a commercial culture system developed for neural differentiation of human embryonic stem cells  as we have previously reported , 24. HumTo further quantitatively characterize the neural specification of hESCs into rosette and NPCs, we have performed quantitative RT-PCR analysis on neural markers . We founin vitro. H1 and H9 hESCs were independently directed to neuronal differentiation in the presence or absence of 20 mM ethanol in biological duplicates. Mixed population of cells pooled from each replicate was harvested at day 12 for neural rosette cells and at day 15 for neural precursor cells (NPC). Gene expression microarray analysis was done using samples from H1 hESC line. Total RNA was prepared and subjected to gene expression microarray as described in Materials and Methods by using Affymetrix Human U133A 2.0 arrays (data accession number GSE56906). We have analyzed the processed data by Weighted Gene Correlation Network Analysis (WGCNA) and also by simple fold change comparisons against control samples.We have used our hESC neural differentiation model to profile gene signatures significantly altered by alcohol exposure, specifically during directed differentiation into neural precursors We have initially performed WGCNA on datasets from both rosette and NPC samples altogether and found that the effects of differentiation  were too overwhelming to see the effect of EtOH treatment on gene signatures (data not shown). Correlation of gene expression to EtOH treatment was not strong enough over the effect of differentiation (data not shown). Therefore, we decided to analyze the dataset for the effect of EtOH on rosette cells separately from on NPC. We performed WGCNA on undifferentiated hESCs (undiff), differentiated without EtOH treatment (EtOH 0 mM) and differentiated with EtOH treatment (EtOH 20 mM). We examined correlations to differentiation and EtOH treatment in neural rosette  or NPC  from theIt was noted that correlation to EtOH treatment was not strong and only a very limited number of modules showed EtOH-dependent association. We reasoned that this was due to the strong effect of neural differentiation on the gene regulation in our model. Next we examined correlative gene expression simply associated with EtOH treatment in differentiated cells. We used datasets from differentiated rosette cells or NPCs with or without EtOH treatment . WGCNA oTo examine potential effects of EtOH on biological process, we have performed DAVID  on genes from the blue , the black (downregulated in NPC), the brown  and the purple (upregulated in NPC). EtOH treatment in neural rosettes showed alterations of genes potentially involved in neuroactive ligand-receptor interaction, cell adhesion molecules and calcium signaling pathway . The blaDHRS3, HNRPD4, THAP2 and ROPN1 demonstrated alcohol induced upregulated expression , a flavonoid component of herbal medicines that counters acute EtOH intoxication [CNTNAP4 and P2RX3 mRNA compared to untreated NPC. The induction was reduced by co-treatment of 1 μM DHM. Furthermore, undifferentiated H9 hESCs and H9 NPC cell lines were treated with 0, 20, and 50 mM EtOH for 48 hrs. From Western analysis, we have confirmed alcohol-induced downregulation of P2RX3 in undifferentiated hESCs and upregulation in hESC-derived NPCs compared to beta-actin or p84 expression . Some of them include: stromal-derived inducing cells, retinoic acid, bone morphogenetic protein, fibroblast growth factor receptors, and neurobasal medium . ESCs carphology . Neural rphology , 32. NPCrphology . DiffereNeural differentiation of ESC and neurogenesis pathway integrates complex epigenetic processes that require strict spatial and temporal cues. Major epigenetic mechanisms including DNA methylation, covalent histone posttranscriptional modifications, chromatin organization, and non-coding regulatory RNA play critical role in pluripotency maintenance and cell fate determination. For insIn relation to this study, epigenetic regulation of the neural transcriptome and the alcohol interference has recently studied and discussed widely. Resendiz et al. have elaborated on the alterations in neural stem cell development and on how alcohol leads to neurodevelopment deficits through neuroepigenetics . As alcoAdditionally, gene expression analysis of hESCs that differentiate into neural cells has facilitated in further defining the molecular mechanisms of neural development. Nestin and βIII-tubulin (TUBB3), which are cytoskeleton proteins, are marker proteins of neural stem cells and neurons respectively . Nestin,in utero has any molecular effect on the onset of the lineage specification of hESCs into neural precursors. Our recent study using hESCs demonstrated significant ethanol-induced molecular alterations in hESCs, especially through epigenetic mechanism [Our study was aimed to assess molecular effect of alcohol on the process of neural differentiation. Potential teratogenic effects of alcohol on fetal development have been documented. Especially studies have demonstrated deleterious effect of ethanol exposure on neuronal development in animal models and on the maintenance and differentiation of neuronal precursor cells derived from stem cells. Molecular and cellular effects of alcohol on neural stem cells and neural progenitors have been demonstrated , 37, 38.echanism .CNTNAP4 and P2RX3. CNTNAP4 is Contactin Associated Protein-Like 4 and belongs to the neurexin family, members of which function in the vertebrate nervous system as cell adhesion molecules and receptors. It is a presynaptic protein involved in both dopaminergic synaptic transmission and GABAergic system, thereby participating in the structural maturation of inhibitory interneuron synapses. Involved in the dopaminergic synaptic transmission by attenuating dopamine release through a presynaptic mechanism. Studies have demonstrated a potential association of CNTNAP4 autism [P2RX3, Purinergic Receptor P2X, Ligand Gated Ion Channel 3, belongs to the family of purinoceptors for ATP. It functions as a ligand-gated ion channel and may transduce ATP-evoked nociceptor activation [In this study, we have generated neural stem cells from pluripotent hESCs and examined global transcriptomic signature changes affected by ethanol treatment (Gene Expression Omnibus under GSE56906), which will provide scientific insight on potential molecular effects of fetal alcohol exposure on neural differentiation of early embryo development . In part4 autism –50 or altivation , 52. It S1 FigA. The level of neural makers  was assessed by qRT-PCR in rosette and NPCs and compared to undifferentiated hESCs. B. The effect of 20 mM EtOH treatment on neural markers  in neural rosette and NPCs was determined by qRT-PCR assay. C. The level of pluripotent markers, Oct4 and Sox2, was assessed in undifferentiated hESC (Und) and NPCs without (0 mM) or with (20 mM) EtOH treatment. Bars are mean ± SEM from triplicates; the asterisk denotes significant (p<0.05) difference from control (one-way ANOVA).(TIFF)Click here for additional data file.


Vicia faba cotyledon culture system. On transfer of cotyledons to culture, their adaxial epidermal cells spontaneously trans-differentiate to a reticulate architecture comparable to their abaxial epidermal transfer cell counterparts formed in planta. Uniform wall layer construction commenced once adaxial epidermal cell expansion had ceased to overlay the original outer periclinal wall on its inner surface. In contrast to the dense ring-like lattice of cellulose microfibrils in the original primary wall, the uniform wall layer was characterized by a sparsely dispersed array of linear cellulose microfibrils. A re-modeled cortical microtubule array exerted no influence on uniform wall layer formation or on its cellulose microfibril organization. Surprisingly, formation of the uniform wall layer was not dependent upon depositing a cellulose scaffold. In contrast, uniform wall cellulose microfibrils were essential precursors for constructing wall ingrowth papillae. On converging to form wall ingrowth papillae, the cellulose microfibril diameters increased 3-fold. This event correlated with up-regulated differential, and transfer-cell specific, expression of VfCesA3B while transcript levels of other cellulose biosynthetic-related genes linked with primary wall construction were substantially down-regulated.Transfer cells are characterized by wall labyrinths with either a flange or reticulate architecture. A literature survey established that reticulate wall ingrowth papillae ubiquitously arise from a modified component of their wall labyrinth, termed the uniform wall layer; a structure absent from flange transfer cells. This finding sparked an investigation of the deposition characteristics and role of the uniform wall layer using a  Transfer cells (TCs) are located at key sites throughout the plant body where they support high transport fluxes of nutrients between apo- and symplasmic compartments. Broadly these sites function in nutrient acquisition across interfaces including soil/root, maternal/filial tissues of developing seeds and host/biotroph or in loading/unloading of vascular pipelines to regulate nutrient partitioning between competing sinks  papillae to repeating fenestrated layers of WI papillae that branch and fuse  morphology and function comparable to their abaxial counterparts  plants raised under controlled environmental conditions. Cotyledons were surgically removed from their seed coats and prepared for aseptic culture on a Murashige and Skoog (MS) medium  were peeled from the cultured cotyledons. The peels, with many cells fractured at their anticlinal walls , strips of epidermal cells (average size 15 mm2O (8:1:2) for various lengths of time to remove their protoplasts and cell wall matrix polysaccharides including hemicellulose  was based on an acid hydrolysis procedure developed by Sugimoto et al.  that remThe epidermal peels were rapidly hydrolysed in the acid bath. Hence a balance was sought between optimizing areas of exposed cellulose microfibrils available for FESEM observation and remaining peel numbers to permit statistical estimates of cellulose microfibril properties. A 5-min acid hydrolysis best met these criteria starting with a batch of 16 peels, with one peel per replicate cotyledon Figure .Cells of cleared epidermal peels (see above) were imaged by FESEM at 100 kx magnification. In randomly chosen fields of view (3.1 × 2.1 μm), in which the cellulose microfibrils of the original wall were apparent, presence/ absence of uniform wall layer cellulose microfibrils was recorded . Each image was overlain by a randomly-positioned grid comprised of 4 × 4 squares (each 600 nm). Numbers of intersections by cellulose microfibrils across the grid lines were scored to provide a relative estimate of their densities. This was repeated 5 times per image and then replicated as described above for estimates of cellulose microfibril coverage.https://mc2.engin.umich.edu/wp-content/uploads/sites/227/2015/11/sputter_coater.pdf) for being applied in an argon atmosphere at a plasma current of 9 mA and applied voltage of 1 kV for 30 s.Image J length selection tool was used to measure diameters of cellulose microfibrils captured in FESEM images of the “cleared” original or uniform wall layers corrected for the thickness of the platinum coating. The thickness of sputtered platinum coating was estimated to be 3 nm according to the manufacturer's instructions (see 2/μm) provided averaged estimates of wall layer thickness. Measures of cell lengths and outer periclinal surface areas were obtained from SEM and confocal laser scanning micrographs of epidermal peels using Image J software  were summed over the total cell numbers per cotyledon. The latter values were deduced from the ratios of cotyledon to cell surface areas . Transcripts were annotated using Blast2GO against NCBI Genbank  corrected P < 5% , NADH dehydrogenase subunit 4 (VfNADHD4), 60S ribosomal protein subunit L2 (Vf60SL2), and multi-domain cyclophilin type peptidyl-prolyl cis-trans isomerase G (VfPPlaseG). The housekeeping genes were further validated using GeNorm analysis of real-time PCR data derived from cDNA of epidermal peels that collectively yielded a reliable reference M < 0.15  were designed using Primer 3 plus  and synthesized by Sigma-Aldrich Australia  known to selectively block deposition of the uniform wall layer  nor stabilizing (taxol) the cortical microtubule array ethane-N,N,N',N'-tetraacetic acid) to dampen the cytosolic Ca2+ signal regulating WI papillae formation ; a relationship consistent with WI papillae construction being dependent on precursor cellulose microfibrils in the uniform wall layer laid down as patches.The percentage of epidermal cells forming WI papillae was fully restored in the DCB and BAPTA recovery treatments Figure . HoweverPotential contributions of enzymes encoded by cellulose biosynthetic-related genes to the observed changes in cellulose microfibril organization and diameters during formation of the uniform wall layer and WI papillae were examined by interrogating a validated RNAseq data set  and KORRIGAN (KOR) and two GPI-anchored proteins, COBRAL-1 and COBRAL-7, that facilitate cellulose biosynthesis  a temporal profile of the volume and rate of deposition of new wall material across the 15-h culture period  Figures . During  Figures . Such a  Figures . Indeed, Figures . Thus, o Figures . Peak co Figures .VfCesA and VfCSL homologs showed that their expression peaked at 1 h of cotyledon culture corresponding with maximal rates of original wall deposition  and VfCSL-D3 , pharmacological disruption of the re-modeled cortical microtubule network in the In the presence of the cellulose biosynthetic inhibitor, DCB  is the recent finding of an increased cellulose microfibril aggregation between primary and secondary walls of VfCesA1, VfCesA3A + VfCesA3B, VfCesA6 approximate the 1:1:1 ratio predicted for a cellulose synthase complex  and (iii) could be met by a temporally graduated change in the CesA composition of cellulose synthase complexes in transitioning from cellulose synthesis for uniform wall layer to WI papillae construction. This transition was characterized by a marked temporal shift in expression levels of genes encoding cellulose biosynthetic enzymes Figures . At 3 h d Figure  so that JP and CO conceived and designed the research project. XX and H-MZ performed the experiments and compiled the data sets and assisted by JP and CO the interpretation of images and analysis of data. XX wrote the first draft of the manuscript that was revised by JP and reviewed by CO, XX, and H-MZ.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Development in mammals is accompanied by specific de novo and demethylation events that are thought to stabilize differentiated cell phenotypes. We demonstrate that a large percentage of the tissue-specific methylation pattern is generated postnatally. Demethylation in the liver is observed in thousands of enhancer-like sequences associated with genes that undergo activation during the first few weeks of life. Using a conditional gene ablation strategy we show that the removal of these methyl groups is stable and necessary for assuring proper hepatocyte gene expression and function through its effect on chromatin accessibility. These postnatal changes in methylation come about through exposure to hormone signaling. These results define the molecular rules of 5-methyl-cytosine regulation as an epigenetic mechanism underlying cellular responses to a changing environment. Here the authors show that a large fraction of the tissue-specific methylation pattern is generated postnatally. These changes, which occur in response to hormone signaling, appear to play a major role in the regulation of gene expression and tissue maturation in the liver. Following implantation, all subsequent changes in DNA methylation appear to be gene specific, with pluripotency genes, for example, becoming de novo methylated at the time of gastrulation3, while tissue-specific regulatory regions undergo demethylation during embryonic development5 or during adult stem cell differentiation6. These processes are triggered by external signaling factors, which then direct interactions between cis-acting sequences and trans-acting proteins within the cell that have the ability to recruit either the de novo methylation complex or demethylation machinery1. Once the epigenetic alterations occur, they serve to autonomously maintain these developmental decisions by generating a stable chromatin template that can persist for long time periods1.DNA methylation patterns are established during mammalian embryogenesis through a programmed process that involves both de novo and demethylation events. Initially, most methyl groups are erased in the early embryo before a new bimodal pattern is established at the time of implantation, where almost all CpG sites undergo de novo methylation while CpG islands are protected by virtue of 7 and stable programmed demethylation of defined regulatory sites also occurs postnatally in the mammary tissue following pregnancy8 as well as in the liver10, where some of these events are mediated by the secretion of testosterone11. It has been suggested that DNA methylation changes may also be induced by the environment or by specific behaviors12. In this paper, we have used high-throughput analysis to demonstrate the existence of programmed postnatal changes in methylation in many different tissues of the body. Furthermore, using the liver as a model system, we show that these changes, which are induced by hormone signaling, are associated with extensive alterations in gene expression that take place during the first few weeks after birth. Finally, by employing genetic intervention, we show that these methylation changes play a role in the process.It is generally believed that the DNA methylation patterns established during development do not undergo further alterations, but recent studies indicate that methylation changes may indeed occur postnatally in terminally differentiated tissues. This has been observed, for example, in cardiomyocytes where many gene regions become hypomethylated or hypermethylated during heart muscle maturation13. By choosing strict criteria to characterize DNA methylation changes (see Methods), we found over 9000 tiles (100 bp) that are undermethylated (>50% reduction) specifically in the adult liver as compared to DNA from a panel of other cell types . While most of these sites are already unmethylated in 5-day-old liver (1 week), about 2800 actually undergo significant demethylation (>35%) postnatally, with a large number of these (~1800) losing their methyl groups only after 3 weeks , a technique that covers a large fraction of the regulatory sequences present in DNA and at sufficient depth to obtain reproducible resultseks Fig. . Unbiaseeks Fig. . Intereseks Fig. . Of note19, thus setting the stage for replacement either through replication dilution or active DNA repair processes23. This also seems to be the case for the postnatal demethylation observed in the liver, as indicated by whole-genome analysis (see Methods), demonstrating that these DMRs are highly enriched for 5hmC  carrying the gene for Cre recombinase under the control of the hepatocyte-specific thyroid-binding globulin (Tbg) promoter27 and followed until they reached the age of 15 weeks. Preliminary control experiments indicated that infection with AAV in this manner works efficiently to delete target sequences . Similar results were obtained when gene ablation was carried out in older animals (15 weeks) following completion of all demethylation events  that allowed the accumulation of highly significant data, revealed approximately 52,000 regions that undergo demethylation postnatally (Supplementary Dataset https://mygga.genomatix.de/) of transcription factor families known to be important in hepatocytes  are associated with genes (n = 1212) that show a significant  postnatal increase in mRNA levels  that are upregulated during liver maturation between 1 and 15 weeks. Moreover, this increase in transcription was significantly inhibited in Tet2/Tet3 double mutant animals  are associated with genes that do not undergo significant postnatal induction , appear to be packaged into nucleosomes containing the enhancer marks H3K4me1 and H3K27ac, are sensitive to DNaseI, and bind liver-specific transcription factors to the same extent as those located within the genes that do undergo postnatal transcriptional activation  on both wild-type (WT) and Tet2/Tet3-deficient (1 day) adult hepatocytes and specifically examined the postnatal DMR regions. Strikingly, while many DMRs were detected in this assay, these same exact sites were found to be locally inaccessible in the mutants Fig. . This is11, as confirmed by showing that these biochemical changes do not occur in castrated mice  and lipid  metabolism possibly explaining the DKO phenotype  and adult (15 weeks) samples. As in the liver, we observed considerable developmentally generated undermethylation in each tissue, but in addition, identified many sites that undergo demethylation postnatally Fig. . In all 1. Recent studies have indicated that most of these DMRs correspond to enhancer-like sequences38 that often act as regulatory elements for nearby genes. It has been assumed that these changes take place exclusively during tissue formation in the embryo, yielding mature cell types that then continue functioning with the same stable methylation pattern for the lifetime of the organism. Here we demonstrate that many tissues continue to shape their epigenetic landscape after birth, suggesting that each cell type may undergo post-developmental molding even after the main process of tissue differentiation has terminated.During development, individual cell types are generated through a serial process of differentiation characterized by programmed changes in gene expression that give each tissue its specific qualities and functions. In mammals, this is accomplished in parallel with alterations in DNA methylation that serve to stabilize the differentiated state39, we generated a conditional Tet2/Tet3 double mutant that completely inhibited the biochemical removal of methyl groups from their programmed regulatory sites in vivo, thus enabling us to directly test the role of demethylation in the regulation of hepatocyte gene expression. Our studies indicate that in the absence of demethylation, many genes are inhibited in their ability to undergo induced expression postnatally and this is observed to a large extent precisely at those genes that contain DMRs, indicating a direct effect. We have also carried out important control experiments showing that the TET proteins themselves have no influence on target gene transcription once they are already demethylated . Our experiments (ATAC-Seq) indicate that removal of methyl groups brings about a local increase in accessibility that promotes additional transcription factor-binding in vivo. Indeed, it is likely that while tissue-specific factors may be responsible for priming these regulatory sequences, demethylation could serve to open them further in order to allow the binding of additional transcription factors. In keeping with this hypothesis, an analysis of data obtained from the human ENCODE project indicates that a variety of different general factors bind exclusively to DMRs in each specific cell type, even though there are many other methylated enhancer-like binding sites present in the same cell  were injected into the superficial temporal vein of 1-day-old mice following anesthetization on ice. Three-week-old mice were anesthetized and injected in the jugular vein with 1012 genome copies of this same virus. Liver-specific insulin receptor knockout mice were generated in a similar manner using InsRF mice34. All animal experiments were performed in accordance with the guidelines of the Hebrew University Institutional Committee for the Use of Animals for Research.C57Bl mice were used for all experiments. DKO mice were generated from Tet246. Cells were then stained with a CD-45 antibody (Biotech) and FACS sorted according to size and to negative staining for CD-45. This yielded a population of relatively pure (>95%) hepatocytes. Unsorted liver cells from newborn mice were seeded and grown on collagenase-coated tissue culture plates containing hepatocyte medium supplemented with 5% fetal bovine serum, 1% hepatic growth supplement, 100 U/mL penicillin, and 100 µg/mL streptomycin (ScienCell) for 2–4 weeks.Mice were anesthetized, sacrificed, and subjected to liver perfusion. Whole liver was then digested by collagenase (Roche) and hepatocytes removed and isolated by means of a percoll gradient13 and run on HiSeq 2500 (Illumina) using 100 bp paired-end sequencing. WGBS libraries were prepared using the Ovation® Ultralow Methyl-Seq Library System (NuGEN Technologies Inc.) as per the manufacturer’s instructions. One hundred and fifty nanograms of genomic DNA was sheared with the M220 Focused-ultrasonicator™(Covaris®) using the 200 bp snap-cap protocol and subsequently purified with AMPure XP beads. After end repair, adaptor ligation, and final repair, DNA was bisulfite converted with the EpiTect Fast DNA Bisulfite Kit (Qiagen GmbH) following the cycling conditions specified in the Ovation® Ultralow Methyl-Seq Library System protocol. Libraries were then amplified, purified, pooled, and sequenced on the Illumina HiSeq with 70 bp paired-end sequencing. Sequencing was conducted by the University of Pennsylvania Next-Generation Sequencing Core  at a depth that exceeds the 30-fold “gold standard” commonly achieved in WGBS databases.DNA was isolated from mouse hepatocytes or from snap-frozen mouse tissues and incubated in lysis buffer (25 mM Tris-HCl (pH 8), 2 mM ethylenediaminetetraacetic acid, 0.2% sodium dodecyl sulfate, 200 mM NaCl) supplemented with 300 μg/mL proteinase K (Roche) followed by phenol:chloroform extraction and ethanol precipitation and RRBS libraries were prepared®) using the microTube snap-cap. Five micrograms of chromatin was then immunoprecipitated with 2 μg of H3K4me1, H3K27ac (Abcam), or HNF6  antibodies. Libraries were generated using the NEBNext Ultra™ II DNA Library Prep Kit for Illumina (New England Biolabs).RNA was isolated from mouse hepatocytes using the miRNeasy Kit according to the manufacturer’s instructions (Qiagen). RNA-Seq libraries were generated using the TruSeq RNA Sample Preparation Kit v2 (Illumina). For ChIP analysis, chromatin was fixed and sheared with the M220 Focused-ultrasonicator™  and subjected to PCR amplification to make the library48. Magnetic beads (AMPure XP) were used to purify an ATAC-Seq library47, followed by quality control with a high-sensitivity DNA bioanalysis chip (Agilent), quantification with KAPA (Illumina), and QuBit. The libraries were sequenced (75 bp paired-end) on Next-Seq (Illumina).The ATAC-Seq protocolFor 5hMeDIP-Seq, DNA was isolated from hepatocytes harvested from a 20-day-old mice and sheared with the M220 Focused-ultrasonicatorTM (Covaris) using snap-cap tubes. Libraries were generated using the NEBNext Ultra DNA Library Prep Kit (NEB E7645). Library DNA was denatured in 95 °C for 10 min, and then the denatured DNA was precipitated with 5hmC Ab (Active Motif 55010) and sequenced (50 bp single-end) on Next-Seq (Illumina).Blood serum was collected by cardiac puncture from overnight-fasted mice and tested for cholesterol, HDL, and triglyceride levels. Blood glucose levels from overnight-fasted mice were measured by glucose test strips (Roche). Liver tissue was fixed in 4% formaldehyde for 8 h and then preserved in 70% ethanol. Liver was embedded in paraffin, sliced, and mounted on slides, which were then stained with an E-Cadherin antibody and Cherry secondary antibody as well as DAPI  (Sigma). Cell size was calculated using the Image J software.A chart of all high-throughput experiments is shown in Supplementary Fig. trim_galore–rrbs–paired sample_R1.fastq.gz sample_R2.fastq.gz49) using the following commands:Read alignment (genome build mm9) and extraction of single-base resolution methylation levels were carried out by BSMAP v.2.74  in newborn (1 week) as compared to adult hepatocytes. This slightly lower cutoff was used to take into consideration the possible loss of methylation that takes place immediately after birth, but prior to our first hepatocyte DNA sample (1 week). The same process was performed for tissue-specific demethylation of other tissues . WGBS was analyzed in a similar manner to the RRBS data. Two hundred base pair DMRs with a minimum coverage of 15 CpGs were identified. In order to find hepatocyte-specific undermethylated tiles at the whole-genome level, we compared our results to a published data set on other tissues (GSE42836). Postnatal tiles were found using similar parameters to those of RRBS.Percent methylation was calculated for 100 bp tiles with a minimum coverage of 10 CpGs. Hepatocyte-specific undermethylated or de novo methylated regions were at least 50% less or more methylated in five out of nine different tissues. This relatively large cutoff was chosen in order to maximize for specificity and significance. Using these DMRs as our starting material, postnatally demethylated regions were then defined as being at least 35% more methylated .The statistical significance of the original tissue-specific DMR set was calculated using a permutation test: The 50). Differential expression was identified by CuffDiff and Cufflink version 2.2.1 (Ref. 51) . These data were normalized by counting fragments per kilobase million units and dividing by the average of all treatments. Our studies also allowed us to look at the overall effects of preventing postnatal demethylation. RNA-Seq, for example, identified a total of about 870 genes that are expressed at lower levels in the DKO as compared to WT 20-week hepatocytes and many of these (n~400) are significantly upregulated postnatally. We also detected 790 genes that are actually upregulated in the knockout as compared to WT. These changes may result from indirect effects downstream of the primary methyl-dependent genes or from activities of the TET proteins not related to DNA methylation52. Alternately, they may come about as a compensation response to changes in physiology resulting from the DKO. Expression in non-hepatocyte tissues was derived from published RNA-Seq data .For RNA-Seq, ChIP-Seq and ATAC-Seq sequences were trimmed with cutadapt v.1.15 and aligned with bowtie2 v.2.2.4. Duplicated sequences were removed with samtools rmdup. RNA-Seq 50 bp single-end reads were obtained from HiSeq 2500 and analyzed by TopHat2 (Ref. https://www.encodeproject.org/), HNF4α (GSM1390711), RXRa (GSM864674), PPARα (GSM864671), and DNaseI (GSM542592). Motif analysis was carried out by HOMER (findMotifsGenome.pl). For ATAC-Seq 75 PE sequences were aligned and duplicated as well as mitochondrial DNA was removed before peak calling by homer.ChIP-Seq and 5hmDIP-Seq 50 bp single-end reads were obtained from HiSeq 2500 and analyzed by Homer  and heatmaps were prepared by Deeptools (computeMatrix). ChIP-Seq distribution was generated by Homer (annotatePeaks.pl). The Genome browser view was generated by converting Bam files into using the bedGraphToBigWig tool and then uploaded on the IGVtools (count). ChIP-Seq data were obtained from the publicly available GEO database, including H3K4me1 and H3K27ac data from the liver, lung, heart, and hippocampus collected from the ENCODE website  were aligned to the mouse genome (mm9) using Bowtie. Aligned reads were analyzed using HOMER, filtering out paired reads that map within 1 kb of each other that map to regions with at least 5× higher than average sequencing coverage or that are not within 500 bp of a The sequencing data have been deposited in the GEO under accession number GSE85251. Accession codes for previously available datasets also used here are listed in the data analysis section. The data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1


Individual prophages and the host pneumococcal genetic lineage were strongly associated and some prophages persisted for many decades. RNA sequencing provided clear evidence of prophage gene expression. Overall, pneumococcal prophages were highly prevalent, demonstrated a structured population, possessed genes associated with virulence, and were expressed under experimental conditions. Pneumococcal prophages are likely to play a more important role in pneumococcal biology and evolution than previously recognised.Bacteriophages (phages) infect many bacterial species, but little is known about the diversity of phages among the pneumococcus, a leading global pathogen. The objectives of this study were to determine the prevalence, diversity and molecular epidemiology of prophages  among pneumococci isolated over the past 90 years. Nearly 500 pneumococcal genomes were investigated and RNA sequencing was used to explore prophage gene expression. We revealed that every pneumococcal genome contained prophage DNA. 286 full-length/putatively full-length pneumococcal prophages were identified, of which 163 have not previously been reported. Full-length prophages clustered into four major groups and every group dated from the 1930–40 s onward. There was limited evidence for genes shared between prophage clusters. Prophages typically integrated in one of five different sites within the pneumococcal genome. 72% of prophages possessed the virulence genes  Streptococcus pneumoniae (the pneumococcus), which is also a major cause of paediatric meningitis and bacteraemia. Children who survive life-threatening pneumococcal diseases often have profound disabilities1Infectious diseases are a leading cause of early childhood deaths, among which pneumonia is the most common: an estimated 1.3 million children died of pneumonia in 2013. The leading cause of pneumonia was Safe and effective pneumococcal vaccines are available to prevent disease, but vaccines are not yet universally administered among all countries, particularly those in the geographical regions in need of the greatest protection. Furthermore, although they are safe and effective, current vaccines are limited in the protection they provide. Antimicrobials can be used to treat pneumococcal infection, but antimicrobial-resistant pneumococci are found worldwide and resistance can result in treatment failures. Therefore, despite the availability of vaccines and antibiotics, pneumococcal disease remains a major global challenge34Phages infect bacteria by attaching to surface-exposed host receptors and injecting their DNA into the cell. There are virulent phages that are only lytic, and temperate phages that can be both lytic and lysogenic. In the lytic cycle, phages synthesise new progeny, cause the host to lyse and release new viruses, while in the lysogenic stage, the viral genome is stably integrated into the bacterial DNA and replicates along with the bacterial chromosome. Prophages  can become lytic in a process called induction, which is triggered spontaneously or by external stimuli such as exposure to mitomycin C. Prophages are one type of mobile genetic element, transferring DNA to and from the bacterial genome6Vibrio cholerae and Staphylococcus aureus), enhancing bacterial adherence to platelets (Streptococcus mitis), or evading immune defences (Pseudomonas aeruginosa)67Streptococcus pyogenes contains six different prophages or prophage-like elements that together comprise approximately 12% of the bacterial genome and have been shown to directly contribute to the high virulence of that lineageExpressed prophage genes often have a phenotypic effect on the host bacterium, for example by producing a toxin that increases bacterial virulence  determine the prevalence of prophage DNA within a large global and historical pneumococcal genome dataset; ii) assess the genetic diversity and molecular epidemiology of prophages among pneumococci isolated over a 90 year period; and iii) investigate associations between these prophage sequences and pneumococcal genetic lineages.The genomes analysed in this study comprised a diverse set of 482 pneumococci recovered between 1916 and 2008 from people of all ages residing in 36 different countries. Pneumococci were recovered from both healthy individuals and those with disease. 91 pneumococcal serotypes, 258 multilocus sequence types (STs) and 94 different clonal complexes  were represented in the dataset .Overall, 100% of the pneumococcal genomes contained coding sequences annotated as being phage-associated. In particular, 689 full-length, putatively full-length and partial prophage sequences at least 2 Kb in length (see Methods for definitions) were identified among 326 (68%) of the 482 pneumococcal genomes . Phage sIn total, we revealed 163 previously unreported full-length or putatively full-length pneumococcal prophages in this dataset. 93 (66 representative examples) full-length prophage sequences were identified and these were 29–60 Kb (median 37.5 Kb) in length . 76 (56 45% (219/482) of the pneumococcal genomes harboured at least one full-length and/or putatively full-length prophage sequence. Among these 219 genomes, 28% (n = 63) were poly-lysogenic, i.e. contained >1 full-length or putatively full-length prophage sequence within the pneumococcal genome.The 66 representative full-length prophage sequences were aligned and the average pairwise identity was only 40.2% (range 20.8–97.4%); however, pairwise comparisons of prophage sequences and an unrooted phylogenetic tree depicted four major prophage clusters and one single prophage . RemarkaThe evidence for genes shared between prophage clusters was limited. Prophages within every cluster possessed genes involved in DNA packaging, the production of the phage capsid, tail and other structural proteins, and genes encoding hypothetical proteins that were identical or highly similar  to the other prophages in that cluster  and 4. PThirteen representative cluster A prophages had an average pairwise nucleotide sequence identity of 83.6%  pairwise identity overall . Cluster17The 93 full-length prophages were consistently integrated in specific locations of the pneumococcal genome. There were five major categories of integration sites, within which the patterns of pneumococcal genes flanking the prophage integrase and amidase genes were consistent . The flaThe integration site was directly associated with the nucleotide sequence of the prophage integrase and the individual prophage clusters . Based uCluster A prophages were split in terms of integrase groups: seven prophages  had a group I integrase and six  possessed a group III integrase. All cluster B prophages possessed an integrase from group II, apart from IPP5, which had a unique integrase. Cluster C prophages mainly had an integrase from group I, apart from four prophages  that had a group IV integrase. One prophage in cluster D (IPP26) had a group I integrase sequence whereas the four others possessed unique integrase sequences. The single prophage IPP24 contained an integrase sequence from group I.pblA and pblB are phage tail protein genes that were first described in S. mitis and were shown to be associated with platelet binding and an increased risk of endocarditispblB was recently shown to be associated with pneumococcal adherence to human lung epithelial cells and increased persistence in the nasopharynx and lung in a murine model of pneumococcal infectionThe phage tail is used for host recognition, attachment and delivery of the phage DNA to the host cell. pblA and/or pblB , although the pairwise sequence identity among the three versions of pblB possessed by these three prophages was only 67.1%. Overall, pblB sequence diversity was high, which was perhaps not surprising since the phage tail interacts with the host. The average pairwise sequence identity was 63.5% among all 47 pblB sequences, although within-cluster sequence identity was higher: clusters B1 (83.6%); B2 (67.3%); B3 (68.0%); B4 (69.6%); and C (79.7%). The sequence variation within pblB led to sequencing challenges associated with this particular gene, i.e. the majority of the putatively full-length prophage sequences in this study had a fragmented pblB and as a consequence, an incomplete sequence assembly. There were no complete matches to the pblB sequence from the murine model study (NTUH-P15 pblB)pblB in this study were 97–98% similar matches to only ~1.2–4.4 Kb of the NTUH-P15 pblB sequence (data not shown).In the current study, only prophages in clusters B and C (71.6% of the 66 unique full-length prophage sequences) possessed /or pblB  and 4. T6A  and CC41/160519A (n = 18), were lineages of pneumococci recovered in South Africa during the 1970–80 s, apart from one pneumococcus in CC41/160519A that was recovered in the USA in 1985. Twelve different prophages were detected among the 17 pneumococci in CC10946A and 15 of the 17 pneumococci each contained two different prophages. Three different prophages were detected among 14 pneumococci in CC41/160519A and four of the pneumococci each contained the same pair of prophages. The pneumococci of CC10946A and CC41/160519A were isolated from both healthy people and those with invasive disease. CC10946A remains a major pneumococcal lineage causing invasive disease in South Africa6A continue to maintain these prophages is currently unknown.Collectively, the 286 full-length and putatively full-length prophages were identified among 56 different CCs and 36 Singletons (unclustered pneumococci) overall. Five or more prophages were identified among the pneumococci representing 17 different CCs . The two18C, 1514, 1803 and 19919A are all globally-distributed lineages of pneumococci  that are prevalent in carriage and disease worldwide2023F and 156/1629V are also globally-distributed lineages  in disease and carriage, but the majority of pneumococci in these CCs harboured one main prophage.CCs 11318C; ≥60 y); IPP12 ; IPP29 ; and MM1 . Serotypes 18C, 1 and 14 have a high invasive disease potentialFurthermore, there was generally a strong association between individual prophages and the host pneumococcal genetic lineage, that is, unique prophages were usually found in one (or one predominant) CC. Nineteen different prophages were found at least three times within the entire dataset and of these, 13 prophages were found exclusively in a single CC . Most no9V−3), which contained two full-length prophages and one partial prophage, was grown in broth culture and mitomycin C was added to prompt prophage induction. PMEN3 culture samples were taken at sequential time points and RNA was extracted and sequenced. The RNA-seq data clearly showed prophage gene expression among all three prophage sequences  for genes that were similar at a sequence level and shared between prophage clusters. This is relevant because one theory of phage evolution is that modular groups of genes are exchanged between different phages; however, that does not appear to be an accurate reflection of evolution among pneumococcal prophagesThe third major finding was that some prophages persisted over long time periods – sometimes relatively unchanged at a nucleotide sequence level. Given the high level of recombination and frequent exchange of genetic material between unrelated pneumococci, this suggests that some form of selective pressure may be acting to maintain the integration of certain prophages within specific pneumococcal lineages. Moreover, the prophages represent large regions of DNA and therefore the maintenance of so much foreign DNA could suggest that the prophages provide a benefit to the host pneumococcus.in vitro experiment. At a minimum, this provided evidence for a functional set of prophage genes and proof-in-principle that RNA sequencing could be a useful experimental tool to understand prophage gene expression. It remains to be shown whether functional phage proteins are produced in vivo, but the potential for RNA sequencing to contribute to our understanding of pneumococcal prophages is evident.Furthermore, RNA sequencing of the prophages found in the reference strain of one globally-distributed, multidrug-resistant pneumococcal lineage clearly demonstrated prophage gene expression in a simple It is also important to note that the full-length prophage prevalence in our study was almost certainly underestimated. Some of the older genomes sequenced by us and others were comprised of many assembly contigs; this made full-length prophage sequence identification very challenging and for this reason we resequenced some of our older genomes to improve sequence quality. We could not do this for all pneumococci, thus will have missed some full-length prophage sequences if they were split over multiple contigs and the presence of other prophage sequences in the same genome meant that correct prophage assemblies could not be guaranteed. DNA sequencing technology and genome assembly tools are continually improving and newer genome assemblies are comprised of only a few contigs, meaning that incomplete prophage sequences will be less frequent and problematic going forward.The pneumococcal genome collection was compiled to capture population-level diversity and uniquely investigate historical pneumococci, but much of the pneumococcal metadata were unknown and thus it was difficult to associate specific prophage(s) and pneumococcal phenotype from this dataset. However, our work provides the foundation for future studies to explore associations between unique prophages and pneumococcal phenotypes among well-sampled genome datasets.Importantly, it will be essential to understand whether the prophage genes with unknown functions are directly contributing to (or strongly influencing) pneumococcal pathogenesis and virulence. Pneumococcal pathogenesis is complicated and whether or not there is a significant prophage contribution is not yet well understood, but the vast array of prophage DNA within these pneumococcal genomes warrants a detailed exploration.The study dataset consisted of 482 historical and modern pneumococcal genomes  describe1617252627® 16 Instrument and Buccal Swab LEV DNA purification kits. Extracted DNA samples were sent to the Oxford Genomics Centre, where libraries were made, genome sequencing was performed on the Illumina® platform, and de novo sequence assemblies were generated using Velvet3032Pneumococci were cultured using standard protocols and DNA was extracted using the Promega MaxwellAn initial screen of the first 336 genomes in the dataset using PHAST resulted in a prophage DNA hit rate of 86% among the pneumococcal genomesThe screening pipeline returned the three best BLAT hits per pneumococcal genome and each of these was manually examined. This resulted in the identification of seven more new prophage sequences, which were added to our reference prophage database and the entire genome dataset was rescreened. The screens also returned hits to >1 full-length prophage sequences in different locations within a single pneumococcal genome, all of which were manually inspected to confirm poly-lysogeny . The screens also frequently revealed >10 Kb highly similar hits to reference prophages, and investigation of some of these long sequence matches revealed that PHAST and our bespoke screens were missing many full-length prophages, so the gene annotation files for all 482 genomes were manually investigated for evidence of prophage sequences. Ultimately, new prophages were defined as those with a nucleotide sequence <98% identical to any other known prophage in the prophage reference sequence database or our study dataset. Extensive cross-referencing and BLAST searching of previously-identified prophages to the many new prophages identified by manual inspection confirmed the presence and novelty of the detected prophages.pblB) such that the last few gene sequences were missing, but these could be found elsewhere in the genome assembly. However, since many genomes contained multiple prophage sequences it was often impossible to reconnect with confidence the two or more prophage sequence fragments bioinformatically, so only the longest contiguous part of the prophage sequence was extracted and labelled as IPPX(n). ‘Partial’ prophage sequences contained a series of contiguous phage-associated genes, may or may not have included an integrase or amidase gene, were between 2–25 Kb in length, and were also labelled as IPPX(n).Altogether, these analyses revealed three categories of prophage sequences. ‘Full-length’ prophage DNA sequences were defined as those that started with an integrase gene, ended with an amidase or lysin gene and were >28 kb in length. ‘Putatively full-length’ prophages were defined as those that started with an integrase and nearly met the full-length criteria; however, there was an assembly gap at the end  was added to the broth culture tubes to a final concentration of 2 μg/ml after 3 h of incubation (OD600 ≥ 0.5). Just prior to the addition of mitomycin C at 3 h and after 4, 5 and 6 h of incubation, respectively, broth cultures were removed from the incubator for processing. A 0.5 ml aliquot was used to measure the absorbance and the RNA was stabilised in the remaining 9.5 ml of broth culture by the addition of 19 ml of RNAprotect Bacteria Reagent (Qiagen). RNA was immediately extracted from the samples using the Promega Maxwell® 16 Instrument and LEV simplyRNA Cells purification kit, following the manufacturer’s protocol.Seven 10 ml tubes of brain-heart infusion broth were inoculated with pneumococcal reference strain PMEN3 and incubated at 37 °C + 5% CORNA extracts were sent to the Oxford Genomics Centre where library preps were made using RNA-Seq Ribozero kits  and sequencing was performed on the MiSeq . The sequenced forward and reverse reads were paired and mapped to reference genomes using Bowtie2 with the highest sensitivity optionHow to cite this article: Brueggemann, A. B. et al. Pneumococcal prophages are diverse, but not without structure or history. Sci. Rep.7, 42976; doi: 10.1038/srep42976 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.