We present a model of biogenic magnetite formation in eukaryotes and hypothesize this genetic mechanism is used by broad forms of life for geomagnetic sensory perception. Countering previous assertions that salmon olfactory tissues lack biogenic magnetite, we determine that it is present in the form of compact crystal clusters and show that a subset of genes differentially expressed in candidate magnetoreceptor cells, compared to nonmagnetic olfactory cells, are distant homologs to a core suite of genes utilized by magnetotactic bacteria for magnetite biomineralization. This same core gene suite is common to a broad array of eukaryotes and the Asgard clade archaea Lokiarchaeta. Findings have implications for revising our understanding of eukaryote magnetite biomineralization, sensory perception of magnetic fields, and eukaryogenesis. Animals use geomagnetic fields for navigational cues, yet the sensory mechanism underlying magnetic perception remains poorly understood. One idea is that geomagnetic fields are physically transduced by magnetite crystals contained inside specialized receptor cells, but evidence for intracellular, biogenic magnetite in eukaryotes is scant. Certain bacteria produce magnetite crystals inside intracellular compartments, representing the most ancient form of biomineralization known and having evolved prior to emergence of the crown group of eukaryotes, raising the question of whether magnetite biomineralization in eukaryotes and prokaryotes might share a common evolutionary history. Here, we discover that salmonid olfactory epithelium contains magnetite crystals arranged in compact clusters and determine that genes differentially expressed in magnetic olfactory cells, contrasted to nonmagnetic olfactory cells, share ancestry with an ancient prokaryote magnetite biomineralization system, consistent with exaptation for use in eukaryotic magnetoreception. We also show that 11 prokaryote biomineralization genes are universally present among a diverse set of eukaryote taxa and that nine of those genes are present within the Asgard clade of archaea Lokiarchaeota that affiliates with eukaryotes in phylogenomic analysis. Consistent with deep homology, we present an evolutionary genetics hypothesis for magnetite formation among eukaryotes to motivate convergent approaches for examining magnetite-based magnetoreception, molecular origins of matrix-associated biomineralization processes, and eukaryogenesis. Magnnum (Ga) –12. Likenum (Ga) . The mecnum (Ga) –17.Presence of magnetite in eukaryotes has mainly been affirmed through magnetic remanence measurements in magnetosensitive species, e.g., honeybees, birds, mice, fish  possess an innate guidance mechanism utilized for long-distance migration and homing to natal rivers  broad FMR spectrum  seen in the electron spin resonance spectrum is different from that reported for linear chains of magnetosome crystals in MTB and rather resembles the FMR spectra of strongly interacting magnetic particle systems  olfactory epithelium appear as uniformly sized and ellipsoid shaped clusters, with each cluster containing a compact arrangement of individual particles. Clusters range in size from ∼200 to ∼300 nm . Our images of magnetite are strikingly similar to those obtained by Diebel et al. .The physical properties of magnetite in salmon olfactory epithelium were characterized using a combination of ferromagnetic resonance spectrum (FMR) and atomic and magnetic force microscopies (AFM/MFM). The FMR analysis, conducted on intact olfactory rosette (OR) tissues A, provid systems . Consist ∼300 nm C–G and a ∼300 nm D marked  ∼300 nm F to MFM l et al.  (see theOncorhynchus tshawytscha) by contrasting transcriptome profiles of magnetic (MAG) and nonmagnetic (NM) olfactory cells and blood and muscle tissues. Briefly, three replicate MAG cell isolation experiments were conducted by dissociating OR cells, followed by collection of MAG cells using a magnet with a pointed tip placed on the outside, upper portion of the sample vial and allowing the NM cells to settle to the bottom of the vial through gravitational forces. The pellet of MAG cells that accumulated inside the vial at the tip of the magnet and NM cells from the bottom of the vial were aspirated and transferred into new vials for messenger RNA (mRNA) isolation. Because of MAG cell scarcity, three to five sets of ORs were combined for each cell isolation experiment. The MAG and NM samples, plus three sets of ORs, blood, and muscle tissues from three additional fish and a set of ORs from a fourth fish , were subjected to Illumina sequencing for transcriptome profiling. Adjusting for false discovery rates (FDRs) < 0.05, this experiment revealed 610 differentially expressed genes (DEGs) more highly expressed in the MAG relative to the NM cell type and considerably greater difference between MAG and blood and muscle tissues . In the latter two cases, >11,000 DEGs were more highly expressed in each binary comparison. Consistent with DEG results, multidimensional scaling plots show well separated clusters of points by tissue type or experimental condition .After confirming the presence of biogenic magnetite in salmonid olfactory epithelium, we then determined candidate magnetoreceptor genes of Chinook salmon , a random assortment of MAG material attached to NM cells could not provide the data observed here.Discrete differential gene expression distinctions observed repeatedly when comparing MAG and NM cell findings in our study are only consistent with the conclusion that salmon olfactory tissue magnetic properties result from the intracellular presence of biogenic magnetite. With macrophages ruled out (Dataset S1). Among the DEGs were proteins involved in “iron uptake and transport”  and “iron ion binding’” , including ferritin. Also present were proteins associated with keywords “actin” , “microtubule” , and “cytoskeleton” . These results are consistent with the production or maintenance of an organelle, possibly one produced through a cellular machinery process that somehow shares commonalities with mitosis and that involves iron.To broadly characterize the molecular functions of MAG cells, we relaxed the threshold FDR < 0.1 and focused on the 1,588 DEGs more highly expressed within the MAG sample contrasted to the NM sample. These candidate genes were overrepresented in 80 Gene Ontology (GO) categories, including anatomical structure and cell maturation/development, mitotic cell cycle, protein modification, protein binding, and bounding membrane of an organelle  to a database of magnetite biomineralization genes (Dataset S2). We found that 11 MTB magnetosome gene homologs (MGHs) are “universally present” (uMGHs) in eukaryotes, defined as having bidirectional Basic Local Alignment Search Tool protein (BLASTp) matches across at least 12 of the 13 animal genomes . Furthermore, 9 of these 11 uMGHs were contained in genome contents of the Asgard archaea clade Lokiarchaeota (Datasets S2 and S3), which shows monophyly with eukaryotes (Dataset S4 and SI Appendix).To examine the hypothesis that genetic mechanisms controlling magnetite biomineralization in prokaryotes and eukaryotes might share common, ancient origins, we compared the genome contents of 13 eukaryotes . MamP contains an iron-binding residue with a role in iron oxidation (Datasets S2 and S3). Those gene clusters are generally present in magnetotactic Alphaproteobacteria (Datasets S2 and S3). Of the remaining five uMGHs, Mad9, 17, 25, 29 are present in genomes of magnetotactic Deltaproteobacteria and Nitrospirae, the latter also containing Man6 , is most parsimonious with a magnetite biomineralization gene transfer to eukaryotes having involved a Nitrospirae ancestor.A previous survey of MTB Nitrospirae and Proteobacteria genomes indicates they share a core set of five MTB magnetosome genes, MamABEKP . The 11 xidation , but thixidation , 31. Thexidation –16, the xidation , 17. We bacteria , 17, 32 bacteria , 16, 33 ing Man6 , 16. At ing Man6 . Thus, pDanio rerio) and Chinook salmon, which amounted to a total of 244 and 367 genes encoding uMGHs. Of those Chinook salmon genes, 332 matched to 181 zebrafish orthologs and corresponding Zebrafish Information Network (ZFIN) gene codes . Consistent with these findings, significantly overrepresented reactome pathways include zinc efflux and compartmentalization by the SLC30 family; signal transduction; laminin interactions; and the anaphase promoting complex/cyclosome, which regulates progression through the mitotic phase of the of the cell cycle  within genomes of zebrafish . This indicates that uMGHs may show up-regulated expression in the MAG cell sample. The 18 genes were distributed across 7 of the 11 “universally conserved” categories and included MamABEK, Mad9, Mad25, and Man6 . The differentially expressed MamE homolog shows an exceptional level of conservation to MTB proteins (Dataset S4).Considering the full repertoire of uMGHs in Chinook salmon, we then examined whether these genes may be engaged with putative magnetite presence in salmonid olfactory cells, and thus biomineralization, in light of the DEG findings at threshold FDR < 0.1. Based on the full repertoire of protein-coding uMGHs in the salmon genome, 12.5 μMGHs are expected to occur by chance in a random sample equally sized to the MAG DEG dataset. We found 18 uMGHs were among the differentially expressed genes, which approaches statistical significance , suggestive of sophisticated membrane remodeling capabilities , 30. Our39Could ancient serial endosymbiosis events explain magnetite biomineralization in complex life forms , 43 Fig? Since tThe importance of endosymbiosis in the evolution of eukaryotic complexity has become firmly established through accumulation of evidence that mitochondria and plastids (double bilayer membrane-bound organelles) evolved from bacteria , 45. A nPrevious searches for candidate magnetoreceptors in dissociated salmonid ORs using a microscope with an applied rotating magnetic field identified cells with magnetic properties . HoweverOur findings are a transformative advance to generate convergent approaches that may illuminate the mysterious “sixth sense” of magnetoreception. Equipped with genomic findings, genetic tools coupled with those of physics, behavior, anatomy, and physiology can be developed to validate associations between candidate magnetoreceptor cells and neural signal transduction. Whether the ancient biomineralization system we nominate here bears a relationship to the numerous other matrix-mediated biomineralization systems found in living organisms , 13 or pTo minimize contamination by nontarget magnetic particles, the tools used for animal termination and dissection were iron-free and nonmagnetic . All tools and labware used for microscopy protocols, if not presterilized, were cleaned in HCl or ultrasonic bath in EtOH. The tools and labware used for magnetic cell isolation/transcriptomics experiments were cleaned in HCl, with the exception of filter tips used in RNA liquid handling. That work was performed inside a hood equipped with a high-efficiency particulate air filter whenever possible, and tools were covered with plastic wrap as a dust preventative measure. All reagents were ultrapure, molecular biology–grade buffers made with Milli-Q water, and powdered  reagents were hydrated in molecular-grade water and filtered through a 0.22-μm membrane using an HCl-cleaned syringe. Fish were obtained from local fish farms/markets or hatchery operations and killed in accordance with European and German regulations or under the authority of permit issued to Oregon State University (ACUP 4421).O. mykiss, n = 10) was isolated bilaterally and frozen for measurement of FMR absorption spectra acquired using an X-band ESR spectrometer , at a microwave frequency of 9.07 GHz, 4-mW input power, and a magnetic field sweep rate of 200 mT/min. For lock-in detection, the applied magnetic field was modulated with a 0.4-mT magnetic field of 100 kHz frequency. Findings were compared to experimentally observed FMR spectra of MTB quantitatively explained using the theoretical model developed in Charilaou et al. . The scanning probe microscope  was equipped with a small, super-sharp AFM/MFM tip attached to a commercially produced cantilever  and a self-made voltage to current converter with a maximum current of 8 A for the employed solenoid. The salmon magnetite sample was visualized in external fields applied normal (z axis) to the sample surface at field strengths of 0.5, 3.5, 7, 15, and 35 mT. The externally applied magnetic fields orients all particle magnetic moments partially or even completely along the field direction. Since the probe magnetization is also partially aligned, attractive magnetostatic interactions between probe and magnetic nanoparticles result. These interactions are specifically measured upon lifting the probe in the MFM mode of operation and they manifest themselves in terms of a dark contrast. In the tapping mode of operation, the oscillating probe is periodically almost in contact with the sample, which results in a topographical image irrespectively of the nanoparticle magnetic configurations. Additional microscope details are available from  was used as a nonmagnetic control. The MTB were obtained from mud samples collected in the Rhin Tortu, Strasbourg, France . Samples were imaged using a Leica TCS SP5 II Laser Scanning Confocal microscope with a 63× oil immersion objective . fn1-43fx was excited at 488 nm and emitted light collected using a 500 long pass filter. DAPI was excited at 405 nm. The reflectance mode option of the confocal microscope was calibrated using the MTB reflectance. Further analysis and image presentation were performed using ImageJ software . Given the scarcity of MAG cells in olfactory tissues, three to five sets of ORs were combined in each experiment to obtain sufficient material for visualization of the magnetic pellet under a dissecting microscope. The magnetic cell pellet was aspirated and placed in a RNase-free vial with ∼20 μL buffer, followed by transfer of an aliquot (∼20 μL) of the nonmagnetic cells to a separate RNase-free vial. All other transcriptome samples—n = 3 muscle, n = 3 blood, and n = 4 pairs of ORs—were individually processed. The fish fork lengths ranged from 10 to 15 cm.Tissues for RNA isolation were sourced from Chinook salmon reared in a single tank at the Fish Research Laboratory, Corvallis, OR . 15 fish were sampled for OR tissues used in magnetic cell isolation experiments, one fish was sampled for OR deep transcriptome sequencing, and three fish were each sampled for muscle tissue, blood, and additional pairs of ORs. Muscle tissue was used as a negative control to rule out potential presence of contaminants during magnetic cell isolation experiments. For olfactory MAG and NM transcriptome profiling, three replicate experiments were conducted by enzymatically dissociating olfactory tissues, then isolating MAG cells by conducting magnetic collection , during which NM cells collected on the bottom of the vial through gravitational forces (In the presence of QIAzol Lysis Reagent (Qiagen), solid tissues (untreated ORs and muscle) were mechanically homogenized and lysed with an electronic mortar and pestle, while blood and dissociated MAG and NM OR cells were homogenized and lysed by pipetting. Total RNA was isolated from lysed materials using a Qiagen RNEasy Mini kit following manufacturer’s protocols. Samples were submitted to Oregon State University’s Center for Genome Research and Biocomputing core facility for messenger RNA isolation, Illumina library preparation, individual indexing for demultiplexing, and sequencing on an Illumina HiSeq2000. Each experimental pair of MAG and NM samples was sequenced in a single Illumina lane using 101 cycles and paired-end protocols, with one lane also including the additional snap-frozen single OR sample for deep sequencing. The other nine samples—blood, muscle, and OR tissues—were single-end sequenced in a single lane using 50 cycles.n = 47,921 transcripts) was selected for inclusion in the reference transcriptome used for read-mapping, differential gene expression analysis, and bidirectional BLASTp comparison to MTB biomineralization proteins (MTB accessions available from Dataset S3).The raw Illumina reads were quality processed with Trimmomatic  version setting having aP values with a threshold cutoff of FDR < 0.05 for broad contrasts between MAG and all tissue types and FDR < 0.1 for analysis of genes differentially expressed in the MAG–NM contrast. Broad relationships among gene expression profiles were visualized and inspected through multidimensional scaling plots (EdgeR function plotMDS) and heatmaps (gplot version 3.0.1 function heatmap.2) generated in EdgeR version 3.12.1 with R version 3.2  orthologs  to identify PANTHER-compatible ZFIN identifiers  and reactome pathways  were assessed on the basis of fold-enrichment values, dividing the observed by expected numbers of per GO or pathway term. This denominator is based on the zebrafish background genome and considers the number of genes in the input file. The zebrafish genome was used as a background genome. Statistical significance was adjusted for multiple tests using PANTHER’s built-in B-H FDR correction function. Individual zebrafish and Chinook salmon genes encoding uMGHs (see below) were also categorized by PANTHER family/subfamily groups and protein classes, based on ZFIN identifiers  were annotated using the Protein ANalysis THrough Evolutionary Relationships classification system   in the ZFIN data repository  to a database of magnetosome proteins of distantly related phyla including Nitrospirae and Proteobacteria  (accessions provided in Dataset S3). An MGH was classified as “universal” (uMGH) in eukaryote genomes if a bidirectional BLASTp match to a named MGH occurred across at least 12 of the 13 eukaryote genomes (>92%), allowing for one missed protein product annotation or gene loss. Nonspecific matches were filtered by applying a threshold cutoff Expect value (E) of E < 1e−3, considered reliable for inferring gene descendants with distant homology (Dataset S3). Genes labeled as “Unknown” (n = 7) were excluded from consideration as uMGHs. To account for evolutionary distance, the Lokiarchaeota uMGH assessment included matches to uMGHs and similarly named MTB homologs.Whether distant homologs of MTB biomineralization proteins are universally present among eukaryote genomes was assessed by comparing genome contents of 13 phylogenetically diverse eukaryote taxa  by the genome-wide number of protein-coding genes . Only DEGs characterized as protein coding were considered in this analysis . Calculations were made in R package stats version 3.2.1 (P < 0.05.The full repertoire of genes encoding uMGHs in zebrafish and Chinook salmon was identified through unidirectional BLASTp queries of fish genome contents to the magnetosome protein database, applying a threshold  matches . As the on 3.2.1 , with stSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary File


In line with the widespread use of this technique, its complications have also received attention.After giving his written informed consent, a 43-year-old man underwent a third session of radiofrequency (RF) catheter ablation for drug-resistant, symptomatic paroxysmal AF at the Jikei University Hospital in April 2018. In the first and second ablation procedure in November 2016 and May 2017, respectively, all 4 pulmonary vein (PV) isolation, cavotricuspid isthmus ablation, superior vena cave isolation (in the first procedure), and additional linear ablations in the left atrium (LA)  were successfully performed. In the index (third) procedure for recurrent AF and atrial tachycardia, additional RF applications were performed to the reconducted roof line, LA bottom line (LA posterior box isolation was established) using a saline-irrigated catheter with a maximum RF energy of 35 watts . During At Saitama Red Cross Hospital, a 40-year-old man with paroxysmal AF underwent PV isolation and a posterior wall isolation procedure using a standard irrigation catheter for the RF application , Table 1At the same hospital, a 44-year-old man with persistent AF underwent PV isolation and a posterior wall isolation procedure using a standard irrigation catheter for the RF application, with a maximum energy output of 25 watts (20 watts in front of the esophagus) , Table 1The patient in case 1 is the only patient among 4000 AF ablations (1/4000) in Jikei University Hospital and cases 2 and 3 are the only 2 cases among 5000 AF ablation cases (2/5000) in the Japanese Red Cross Saitama Hospital in which the intractable diarrhea appeared following the catheter ablation of AF. Therefore, the estimated frequency of this complication will be 0.025% to 0.04%. None of the 3 cases demonstrated the bradycardia/hypotension or vagal responses during the box isolation procedure.,This report describes a lesser-known adverse effect—persistent diarrhea—of catheter ablation of AF. This probably results from the effects of RF energy delivered to the left atrium on the periesophageal vagal plexus. Although upper GI complications after catheter ablation of AF, such as esophageal damage/fistula, and gastric hypomotility, are well known,The clinical presentations of all 3 patients were very similar and the common features of these 3 cases were as follows: (1) all were relatively young men with a good physique, (2) the patients underwent box isolation by RF ablation using an irrigation catheter, (3) diarrhea appeared 2–3 weeks (10–21 days) after the ablation procedure, (4) persistent severe (watery) diarrhea lasted 3 months to 1 year, (5) all patients showed a large amount of weight loss (5–15 kg), (6) hypokalemia was present, (7) there was an absence of marked elevation of the inflammatory response (C-reactive protein), and (8) diarrhea gradually responded to ramosetron or Tsumura Kampo Rikkunshito extract granules and polycarbophil calcium.,Although there have been various reports on the complications of AF ablation, diarrhea is generally not included in the list of complications.Different from the catheter ablation of arrhythmias, diarrhea is a well-known complication after laparoscopic antireflux surgery or paraesophageal hernia repair.The progression of hypokalemia is one of the striking features of our patients, especially in the first case. The patient developed severe hypokalemia with quadriplegia as a consequence of persistent watery diarrhea and lost 15 kg of body weight, despite receiving potassium replacement therapy. We should realize that diarrhea and abnormal bowel movement should not be overlooked and should be treated carefully as a possibly fatal complication of catheter ablation for AF.All 3 cases in this paper underwent box isolation with the index procedure, which isolates both pulmonary veins and the left atrial posterior wall . Both th•Although upper gastrointestinal complications after catheter ablation of atrial fibrillation (AF), such as esophageal damage/fistula and gastric hypomotility, are well known, less is known about lower digestive tract symptoms, including diarrhea.•The common clinical presentations of cases with intractable diarrhea are as follows: relatively young patients with a good physique, box isolation by radiofrequency ablation using an irrigation catheter, diarrhea appeared 2–3 weeks after the ablation procedure and lasted 3 months to 1 year, large amount of weight loss (5–15 kg), and hypokalemia .•Diarrhea and abnormal bowel movement should not be overlooked and should be treated carefully as a possibly fatal complication of catheter ablation for AF.It is noteworthy that diarrhea appeared after a substantial period (10–21 days after the procedure) in all cases. Both the patient and the attending physician may be unable to realize that diarrhea is a complication of the ablation procedure and the patient is likely to undergo gastroenterological examinations to investigate possible hidden diseases, such as inflammatory bowel disease. This is the first report demonstrating that persistent diarrhea is a possible complication of left atrial catheter ablation. Awareness and recognition of this complication may result in a better understanding of the magnitude and clinical spectrum of this complication.


Cellular senescence is a process that can prevent tumour development in a cell autonomous manner by imposing a stable cell cycle arrest after oncogene activation. Paradoxically, senescence can also promote tumour growth cell non-autonomously by creating a permissive tumour microenvironment that fuels tumour initiation, progression to malignancy and metastasis. In a pituitary tumour known as adamantinomatous craniopharyngioma (ACP), cells that carry oncogenic β-catenin mutations and overactivate the WNT signalling pathway form cell clusters that become senescent and activate a senescence-associated secretory phenotype (SASP). Research in mouse models of ACP has provided insights into the function of the senescent cell clusters and revealed a critical role for SASP-mediated activities in paracrine tumour initiation. In this review, we first discuss this research on ACP and subsequently explore the theme of paracrine tumourigenesis in other tumour models available in the literature. Evidence is accumulating supporting the notion that paracrine signalling brought about by senescent cells may underlie tumourigenesis across different tumours and cancer models. The al aging . HoweverINK4/Rb and p21CIP1/p53 pathways, the activation of anti-apoptotic mechanisms and the widespread secretion of growth factors, cytokines, chemokines and extracellular matrix components (collectively known as the senescence-associated secretory phenotype or SASP). The different types of senescent phenotypes and their underlying mechanisms have been thoroughly reviewed elsewhere [The early concept of cellular senescence has now been expanded to describe a growing list of phenotypes initiated by damaging stimuli such as telomere attrition, ionizing radiation, chemotherapeutic compounds, reactive oxygen species (ROS), mitochondrial dysfunction and oncogenic signalling . ImportaSenescent cells and the SASP can induce a vast array of context-dependent effects, playing significant roles in the regulation of normal tissue physiology but also in disease. Senescent cells can be found in several tissues during embryonic development and participate in the proper patterning of some organs and tissues –9. AfterIn the case of cancer and neoplastic diseases, senescence can be induced cell autonomously by oncogene activation  or through therapeutics such as DNA-damaging chemical compounds and ionizing radiation , which lead to the activation of DNA-damage pathways and the activation of a stable cell cycle arrest . AdditioThe cell non-autonomous origin of some tumours stands in stark contrast to traditional models of carcinogenesis –49. A reCraniopharyngiomas (CPs) are benign epithelial tumours (WHO grade 1) of the sellar region, which is an anatomical structure located between the hypothalamus and the cranial base. CPs represent between 1.2 and 4.6% of all intracranial tumours, with an incidence of 0.5–2.5 new cases per 1 million population per year , 51. TheACPs represent the most common non-neuroepithelial intracranial tumours in children and young adults , 53. TheThe histomorphological features of ACPs are well defined Fig. . The tumCTNNB1, the gene encoding for β-catenin, are the most common molecular alterations associated with ACP tumourigenesis [CTNNB1 mutations have been identified in all of the epithelial tumour cells in a large cohort of ACPs by combining laser capture microdissection with deep sequencing [CTNNB1 are tumour drivers and provided important insights into the role of the nucleocytoplasmic β-catenin cell clusters in ACP tumourigenesis (see below) [Molecularly, ACPs are driven by the overactivation of the WNT/β-catenin signalling pathway , 69. Thiigenesis , 69. Theigenesis . In agreigenesis . Despitequencing . Three-dquencing . Importae below) , 75. Thee below) , 76, 77.e below) .Fig. 2ScHesx1Cre/+; Ctnnb1lox(ex3)/+ mouse line) or in SOX2 + adult pituitary stem cells /+ mouse line)  allele, which then codes for a mutant version of β-catenin missing several important regulatory amino acids located in the N-terminal end of the protein. Degradation of this mutant β-catenin is impaired, as the protein cannot be phosphorylated by its destruction complex but can still activate transcription of target genes  in the developing (embryonic) or adult (inducible) pituitary leading to the formation of tumours resembling human ACP [Two genetically engineered mouse models of ACP have been developed by expressing oncogenic β-catenin in either HESX1 + embryonic precursors of the developing pituitary  being present throughout the developing pituitary cells as shown by laser capture microdissection and PCR [In the embryonic model, the expression of oncogenic β-catenin is driven into early precursors of Rathke’s pouch (RP) expressing the homeobox transcription factor Hesx1 . RP is tHesx1 . SporadiHesx1 . AnalysiHesx1 . It is pHesx1 , 82–85.  and PCR . Mouse t and PCR .Hesx1-expressing undifferentiated precursors, clusters do not form and tumours never develop [bona fide organ-specific stem cells results in the initial formation of β-catenin-accumulating cell clusters and subsequent development of ACP-like tumours after a latency period of 3–6 months , an approach known as genetic lineage tracing . Geneticion Fig. a. These ion Fig. b–d and DCIP1); (3) exhibit DNA damage and activation of a DNA damage response; (4) have an enlarged lysosomal compartment ; (5) activate the NF-κB pathway and a SASP [Molecular profiling and genetic approaches have shown that these β-catenin accumulating clusters are enriched in senescent cells in both mouse and human ACP. Specifically, human and mouse cluster cells exhibit several hallmark features of senescence: (1) they are viable and non-proliferative (Ki67 and EdU negative); (2) express cell cycle inhibitors  vs. young (4–6 weeks old) mice. This reduction in tumour burden is associated with a significant decrease in the senescence/SASP response in the clusters of the aged mice. Specifically, pituitaries from older mice contain smaller clusters and reduced expression of SASP factors, while reduced numbers of proliferating cells are also observed surrounding the clusters .Apc (adenomatous polyposis coli)  endothelial-like cells expressing SOX9  Fig. , also le factors . In addiOX9 Fig. c. Therefnduction .In human ACP, the location of the senescent clusters within the finger-like tumour protrusions invading the brain suggests that SASP activities may promote epithelial cell proliferation and invasion . This isParacrine tumourigenesis and senescence-mediated paracrine tumourigenesis mechanisms have not yet been described in other pituitary tumours. In the case of pituitary adenomas, cellular senescence has been shown to restrict tumour cell proliferation and is thought to underlie the almost invariable benign nature of pituitary tumours –97. TherCurrent and widely supported theories of carcinogenesis such as the cancer stem cell/hierarchical model, the genetic/stochastic evolution model and more recent unifying propositions imply that a tumour  is formed by cells that are descendants of the cell originally targeted by an initiating oncogenic insult –49. On tAround 3 decades ago, in vitro experiments had already shown that paracrine or autocrine exposure of naïve cells to secreted developmental growth factors of the WNT and FGF families could initiate pre-malignant phenotypical changes, including: (1) ability to grow without attachment in agar plates (a sign of cell transformation); (2) alteration of cell morphologies; (3) increased proliferation and higher cell culture densities; (4) loss of contact inhibition of proliferation –109. HowDicer1 or the activation of oncogenic β-catenin, specifically in osteoprogenitors or osteoblasts, leads to the development of myelodysplastic syndromes and acute myeloid leukaemias in a paracrine manner. Interestingly, they show that the tumour cell of origin does not carry the targeted mutations (i.e. Dicer1 or β-catenin) and instead display novel mutations and genomic aberrations [In vivo, combinations of different methods such as lineage tracing, cell ablation, DNA sequencing, laser-capture microdissection, chimera aggregations and cell transplantation have been instrumental in providing evidence of paracrine tumourigenesis; while it is important to note that these have also been used to support the validity of cell autonomous mechanisms in some in vivo cancer models –116. Forrrations , 118.N-nitrosomethylurea (NMU) for the induction of mammary epithelial tumours [In breast cancer studies, most experiments so far have used in vitro cocultures to show the capacity of paracrine WNT signalling to transform mammary epithelial cell lines –107. How tumours .In regard to brain cancer, lineage tracing and transplantation experiments have demonstrated an important role for paracrine PDGFB signalling in the recruitment of untargeted oligodendrocyte host cells to induce the formation of gliomas , 122. SiO-tetradecanoylphorbol-13-acetate (TPA) [In skin models of cancer, it is known that certain oncogenic driver mutations require wounding-derived inflammatory signals to initiate tumourigenesis , 131. Inte (TPA) , 134. Fite (TPA) –137. Theinitiation of tumours (as it occurs in mouse ACP models) has been less explored, possibly due to the inherent difficulty in studying the early stages of cell transformation and tumour initiation in vivo.There is ample in vitro and in vivo evidence demonstrating the tumour promoting activities of cellular senescence on cells carrying oncogenic mutations (see reviews cited above). On the other hand, the requirement of senescence and SASP signalling for the cell non-autonomous D. melanogaster. In this model, elegant use of gene targeting, genetic lineage tracing and cell ablation approaches have shown that activating oncogenic Ras induces senescence-like phenotypes and SASP activation in targeted cells, which lead to tumour growth in neighbouring wild type cells through the activation of developmental pathways and JNK signalling [Although a considerable number of in vitro experiments have shown that conditioned media from senescent cell cultures can enhance the cancerous properties of preneoplastic or neoplastic cells (see the reviews mentioned above), it has also been shown that some cells require exposure to senescent cell-conditioned media to acquire cancer stem cell-like phenotypes and the ability to form tumours when transplanted into nude mice –140. In gnalling –144. In gnalling . This, tgnalling –149, impINK4A-expressing senescent keratinocytes and their SASP induce hyperproliferation and activation of WNT signalling target genes in neighbouring naïve cells, leading to the formation of papillomas when DMBA/TPA is applied [In mice, the injection of senescent HRasV12-expressing keratinocytes can induce the formation of skin papillomas that are very similar to those in DMBA/TPA models. Strikingly, a considerable portion of cells within the papillomas contain non-targeted host cells, suggesting an important role for senescence-driven paracrine signalling in tumour formation . Notably applied . These f applied –137.There is also strong evidence suggesting that senescence and the SASP can drive paracrine tumourigenesis in the liver. For example, it has been shown that obese mice develop higher levels of the microbial metabolite deoxycholic acid (DCA) which leads to higher number of senescent hepatic stellate cells (HSCs) and increased SASP expression. Interestingly, depletion of either the senescent HSCs or knockout of IL1B signalling (a common SASP component) significantly prevents the growth of DMBA-induced hepatocellular carcinomas . MoreoveThe evidence discussed above suggests three conceivable mechanisms, acting alone or in combination, that could mediate senescence-mediated paracrine tumourigenesis: (1) induction of proliferation in neighbouring cells, therefore, promoting genomic instability and the appearance of novel mutations , 156; , NOTCH1 mutations are considerably underrepresented in oesophageal cancers, suggesting these are more likely to evolve from epithelial cells without NOTCH1 mutations. In contrast, TP53 mutations, which are several-fold less frequent in the aged oesophagus, are almost universally present in oesophageal cancers, suggesting that these cancers originate from the small fraction of TP53 mutant cells [NOTCH1 to be as abundant as TP53. More recently, the mechanisms of action of known carcinogens have been challenged. By analysing the mutational signatures of tumours generated in mice exposed to one of 20 carcinogens, it has been found that most of these agents are not directly mutagenic on the genome (i.e. they do not increase mutation burden in the tumours), with most mutations, including driver mutations, resulting from tissue-specific endogenous processes. This suggests that these carcinogens promote tumour initiation in a different manner, possibly by creating a permissive environment that allows tumour growth rather than, as initially believed, by increasing mutation rate [Early studies showing a negative correlation between senescent cell burden and tumour progression towards malignancy helped establish cellular senescence as a tumour-suppressive mechanism. However, when viewed under the precepts of widely accepted cell autonomous paradigms of tumorigenesis, this inverse senescence-cancer progression relationship implies that cells carrying oncogenic driver mutations have to either bypass or escape the senescent phenotype for tumours to progress. Although there are some convincing experiments in vitro , 177, cont cells , 181. Thion rate . TherefoThe ACP models have revealed that the relationship between oncogenic driver-mutations, tumour cells-of-origin and the tumour microenvironment (TME) can be much more complex than what can be explained by conventional models of carcinogenesis. Lineage tracing has shown the otherwise counterintuitive observation that tumours can be formed from cells that are not related to the original oncogenic insult. Molecular studies in these models indicate that senescent cells bring about a pro-tumourigenic TME that induces tumour initiation in a paracrine manner through the SASP, while in human ACP, the evidence strongly supports a role of senescent cells in tumour growth and invasion. Further preclinical research using senolytics in the ACP mouse models may pave the way to clinical trials to evaluate the clinical relevance of senotherapies, alone or in combination, against human ACP.It remains to be seen how common the occurrence of senescence-induced paracrine tumourigenesis in other tumours and cancers is, but it will be particularly interesting to assess the role of senescent cells and the SASP in the cancer models for which cell non-autonomous initiating mechanisms have already been described Sect. . Evidenc


Salmo salar), a species of major economic and conservation significance. We show that adult captive exposure further induces differential methylation in an F1 generation that is associated with fitness-related phenotypic differences. Some genes targeted with differential methylation were consistent with genes differential methylated in other salmonid fishes experiencing early-life hatchery rearing, as well as genes under selection in domesticated species. Our results support a mechanism of transgenerational plasticity mediated by intergenerational inheritance of DNA methylation acquired late in life for salmon. To our knowledge, this is the first-time environmental variation experienced later in life has been directly demonstrated to influence gamete DNA methylation in fish.An epigenetic basis for transgenerational plasticity in animals is widely theorized, but convincing empirical support is limited by taxa-specific differences in the presence and role of epigenetic mechanisms. In teleost fishes, DNA methylation generally does not undergo extensive reprogramming and has been linked with environmentally induced intergenerational effects, but solely in the context of early life environmental differences. Using whole-genome bisulfite sequencing, we demonstrate that differential methylation of sperm occurs in response to captivity during the maturation of Atlantic Salmon ( We further tested the hypothesis that adult rearing environments influence offspring methylation patterns and that these differences influence offspring phenotypes by creating pure-type crosses of SAS and wild adults and rearing the offspring in a common environment. We characterized growth-related phenotypic differences in 10-month-old juveniles as well as DNA methylation patterns in their livers, to determine the presence of inherited DNA methylation patterns and their potential influence on proxies of fitness.In this context, the goal of our study was to test the hypothesis that environmental differences experienced by adults during maturation alter DNA methylation of gametes. Because the zebrafish methylome is reprogrammed to the paternal state  juveniles . Water temperature ranged from 5°C to 7°C except during the months of May to September where heat-exchangers were used to extract heat from surface water (Stewart Brook) to warm well-water to between 10°C and 15°C tracking the typical summer increase in temperature. SAS fish were initially fed chopped frozen krill for one month and gradually weaned onto standard hatchery pellet food  by coating it with puréed krill for an additional 3–4 weeks before feeding solely pellet food. Densities increased throughout the study as fish grew but never exceeded 10 kg/m3.Wild Atlantic Salmon  adult Atlantic Salmon returning to spawn in the Miramichi River were collected by seining in September of 2017 and 2018 as a part of regular brood-stock collection program by the Miramichi Salmon Association staff from selected pool habitats in the upper reaches of a major branch (the Little Southwest Miramichi River) of the Northwest Miramichi River . Adult fN = 8 crosses each). All SAS males and females had spent two winters in the hatchery. All wild males and one female were one sea winter fish (1SW) and the remaining females were 2SW fish. Fertilized eggs were incubated in flow-through troughs until first feeding after which offspring from multiple families from each treatment were mixed and transferred to one of three 5000 L square tanks. Fish were fed to satiation on hatchery pellet food until they reached a size of approximately 1.5 g . Egg sizes did not differ between wild and SAS adults  and survival to the eyed-egg stage was over 90% for all but one SAS family that averaged only 47% survival across three replicate egg baskets (D. Roth unpublished data).In 2017, both SAS and wild adult fish were haphazardly netted from holding tanks, gametes were collected, and eggs were artificially fertilized to create pure-type breeding crosses  to conduct whole-genome bisulfite sequencing.Juvenile offspring fish were netted haphazardly from the tanks, euthanized in an overdose solution of eugenol , weight and length measurements taken, and liver tissue was dissected and preserved in RNAlater. We chose liver tissue because it is an organ known for its important role in regulating growth and metabolism e.g., . Juvenili.e., 2018), we collected sperm samples from eight wild and eight SAS males. All wild males were grilse having spent one winter at sea, while all SAS males had spent two winters in the hatchery. Sperm was collected in 2-mL tubes and stored on ice for between 2 and 6 h. Sperm (250 μL) was centrifuged at 7000 rpm for 10 min, the supernatant discarded, and isolated sperm cells preserved in 1.5 mL of RNAlater. While this experimental design does not allow direct inference of parent-offspring transmission of DNA methylation, it does still allow us to infer intergenerational transfer of DNA methylation due to the fact that we controlled the rearing environment for the juveniles (common garden) and thus these fish only differ by their respective parents’ environmental exposures. Because the 2018 fish originated from the following cohort as the 2017 parents, this also allows a degree of repeatability of the effects to be tested.Gamete samples were unavailable from the adults used in the breeding crosses in 2017. The following year (N = 16) and liver (N = 16) genomic DNA samples at the McGill University—Génome Québec Innovation Centre using NEBNext Ultra II DNA library prep kits. Each library was sequenced to an expected depth of 15X using 150 bp paired-end sequencing on a half lane of the Illumina HiSeqX platform (Supplementary Table S1). Raw sequencing reads were trimmed using fastp v0.19.9  and sequenced them as a pool in one lane of an Illumina HiSeqX. Raw sequencing reads were trimmed using fastp v0.19.9 and then aligned to the Atlantic Salmon genome with bwa mem  polymorphisms and removed these sites from the methylation results using bedtools v2.26.0  representing two SAS and two wild fish was poor and thus these samples were excluded to reduce biases in methylation estimates due to low coverage for these samples. The minimum coverage filter ensured that differences in methylation were not due to spurious differences in coverage between groups and the maximum coverage filter removed highly repetitive regions where the confidence in mapping accuracy is low. Differential methylation of CpG cytosines (DMC) was determined using beta-binomial models implemented in the DSS v2.32.0 package  for at least 12 of 16 juveniles or eight of 12 adults. The sequencing performance for four adult samples  R package. We tested for enrichment of biological functions for genes associated with DMRs using Fisher’s Exact Tests and the ‘weight01’ . We first summarized methylation in nonoverlapping 100 bp windows across the genome. Windows required a minimum of three CpG sites and we only retained windows with among-sample variances greater than 0.05 to reduce computational burden when constructing the network . We calculated connectivity between all pairs of regions using the bi-weight midcorrelation raised to the power of 18 to approximate a scale-free network as implemented in the WGCNA package in R  and gene significance (region correlation with phenotype) between DMR-overlapping and non-DMR-overlapping regions using t-tests in R.We used a network-based approach to investigate associations of correlated methylation signatures with juvenile phenotypes . Juvenile individuals were chosen to maximize the number of successfully called genotypes. We required a minimum depth of coverage of 8X to call an individual genotype and we retained only SNPs with a successful genotyping rate of 80% . We further removed SNPs with minimum allele frequency of 5% . We used AMOVA implemented in pegas v0.13  context with greater than 5X coverage, but less than 20X coverage in 75% (9/12) of samples. Mean coverage per individual was 8.3X . CpGs in salmon sperm were highly methylated (average methylation: 89%) and exhibited a bimodal distribution, with ∼5% of CpGs nearly devoid of methylation (<5%) and 91% of CpGs having methylation levels >80% , consistent with levels reported in other salmonids  identified between adult SAS and wild salmon sperm that were grouped into 284 differentially methylated regions . DMRs overlapped 237 genes or their cis-regulatory contexts . Gene ontology enrichment analysis revealed DMRs in sperm were associated with genes significantly enriched  for a variety of functions in signal transduction pathways, brain development, tissue differentiation, muscle development and contraction, and chromatin silencing (Supplementary Table S4).We identified differential methylation for individual CpGs between wild and SAS adults using beta-binomial models . While F1 wild fish were smaller on average, they had similar condition factors .To investigate the potential for inherited methylation patterns in juvenile liver tissues, we quantified DNA methylation at over 23.1 million CpG sites covered with a minimum of 5X and a maximum of 20X in 75% (12/16) of F1 wild and SAS juveniles. Mean coverage per individual was 9.4X . In contrast to sperm, the more metabolically active liver tissues exhibited average methylation levels of approximately 80%. Juvenile liver tissue also exhibited a bimodal distribution of methylation where ∼5% of CpGs were unmethylated (<5%), 76% of CpGs with methylation >80%, and a larger fraction of sites with intermediate methylation levels  between wild and SAS juvenile offspring were organized into 346 DMRs that ranged in size from 51 to 2131 bp, contained between 4 to 40 CpGs, and covered 98% of DMCs with FDR < 0.05 . The average magnitude of methylation differences between SAS and wild offspring were comparable . DMRs in juvenile liver tissues overlapped 274 genes or their cis-regulatory context (Supplementary Table S6). Over-represented biological functions of these genes reflected nervous system development and regulation, muscle development and contraction, signal transduction pathways, and immune system processes (Supplementary Table S7).Differentially methylated CpGs (P < 0.001). The shared regions exhibited the same direction of differential methylation in both tissues  and hierarchical clustering of these regions by individual largely recapitulated the SAS vs wild groupings (GRM4-l) had regions that were in close proximity (<5000 bp). The DMRs in proximity to GRM4-l were hypo-methylated in adult SAS sperm and hyper-methylated DMR in juvenile SAS liver tissue .Despite the fact that the adults we sequenced are not the parents of the juveniles we sequenced we found overlap of DMRs between the two tissues and life stages , we used a network-based approach  with among-individual variances greater than 0.05 . We identified 124 modules that included a total of 4179 genomic regions. Eighteen modules exhibited significant correlations with at least one phenotype . Modules were enriched for a variety of signaling pathways and developmental processes relevant to the correlated phenotypes . The strongest association existed between the “navajowhite1” module and condition factor . The regions in this module were in proximity (5 kb) to genes enriched for mesoderm development and regulation of transcription (Supplementary Tables S5 and S6). In particular, the gene encoding insulin-like growth factor I (IGF-1), a hormone produced in the liver and secreted into the circulatory system, that is a key regulator of growth in muscle and skeletal tissues . Three modules exhibited significant overlap with DMRs identified between juvenile SAS and wild salmon . DMR-overlapping regions in these three modules had consistently higher module centrality than non-DMR-overlapping regions .As a means to investigate whether methylation influences juvenile phenotypic variation . Using outlier detection algorithms, we did not detect significant shifts in allele frequencies between SAS and wild salmon using BayeScan or a polygenic framework , and we only identified two outlier SNPs with FDR < 0.01 in OutFLANK , suggesting an overall absence of differential selection at the genome level between SAS and wild salmon.To account for the possibility of selection causing genetic differences between juvenile SAS and wild salmon and explaining the observed phenotypic differences, we also genotyped individuals for 974,219 single nucleotide polymorphisms  using the aligned bisulfite sequencing reads and performed outlier tests. Overall, we failed to find support for a genome-wide average Fi.e., growth and maturation in a natural vs hatchery setting) experienced after approximately 2 years of common rearing in their natural riverine environment alters both DNA methylation in salmon sperm as well as DNA methylation of hatchery-produced progeny with SAS parents. The maturation-environment effect we demonstrated strongly suggests, as other have reported , the methyltransferase responsible for the addition of new DNA methylation, have been retained following successive genome-duplication events  for salmonid and other teleost fishes (Oncorhynchus mykiss), certain DMNT3 ohnologs are expressed in spermatozoa during late spermatogenesis  indicates it does undergo global methylation reprogramming , and whose life histories involve limited parental care  or stochastic processes . Several lines of evidence suggest the patterns we observed originate at least partly from deterministic processes. First, if methylation changes were completely stochastic, we would expect no bias in the direction of methylation changes. In contrast, our results show that differential methylation in SAS fish is strongly biased (about twofold) toward hypomethylation in both sperm and juvenile livers. Second, despite the fact that our juveniles and adults originated from different cohorts and that juvenile livers will have undergone tissue-specific methylation reprogramming from the state observed in sperm, we detected more DMRs in common between the datasets than expected by chance. Finally, the regions in phenotypically correlated methylation networks that overlap SAS vs wild DMRs are significantly biased toward being centrally located regions in the co-methylated networks providing further indications that these are not random associations.There is an unresolved question of whether epigenetic differences arising as a result of hatchery exposure occur due to deterministic processes  was differentially methylated in SAS sperm cells and has previously been identified as differentially methylated in sperm of Atlantic Salmon reared in a hatchery from fertilization  or liver . Glutamate receptors are well-known targets of selection in many domestic animals  associated with synaptic genes, including multiple glutamate receptors  directly relevant to the phenotypes being studied. In particular, the methylation module in juvenile livers with the strongest phenotypic correlation  contained regions overlapping IGF-1 which is a hormone produced and released from the liver in response to growth hormone (GH) signaling that plays a key endocrine role mediating growth and differentiation of muscle and skeletal tissues and therefore body size  suggesting that hatchery-environment-induced methylation-mediated behavioral changes  could have consequences for the growth trajectories of offspring. We also identified modules  and differential methylation of genes not included in methylation modules that lie downstream of genes in important phenotype-associated modules . Phosphatidylinositol-mediated signaling  and PI3KR1 in particular plays a key role in modulating the response to IGF-1 stimulation , our comparisons may be biased. However, the genomic distribution and density of regulatory relevant CpGs in nonmammalian vertebrates fundamentally differs from that of mammals  exhibited more hypo-methylation relative to wild fish , in contrast to previous work that has demonstrated predominantly hyper-methylation of fish produced and reared in hatchery from the egg stage e.g.,  and thusLike previous studies, we have demonstrated a lack of genome-wide sequence differentiation between hatchery-reared and wild fish , selectii.e., small RNAs) are also well known to mediate intergenerational effects , differential methylation analyses (https://github.com/kylewellband/methylUtil), C/T polymorphism characterization (https://github.com/kylewellband/CT-poly-wgbs). Phenotypic data for juveniles are included in the Supplementary File S1. Supplementary material is available at figshare: https://doi.org/10.25387/g3.14674692.The processed methylation datasets are available in NCBI’s Gene Expression Omnibus (GEO Series accession: GSE162129) and the raw pool-seq and bisulfite-seq data are available at NCBI . The ICSASG_v2 Atlantic Salmon genome is available from NCBI (RefSeq accession: GCF_000233375.1). Code to process the data and generate the results is available on github as follows: generation of methylation counts from sequencing data (


To clarify characteristic angiographic features and clinical efficacy of selective transarterial embolization (TAE) of retained placenta with abnormal vaginal bleeding.The study cohort comprised 22 patients  who underwent selective TAE for retained placenta with abnormal bleeding between January 2018 and December 2020 at our institution. Angiographic images were reviewed by two certified radiologists with consensus. Medical records were reviewed to evaluate the efficacy of TAE. Angiographic features of retained placenta, technical success , complications, clinical outcomes (hemostatic effects and recurrent bleeding) were evaluated.Pelvic angiography showed a dilated vascular channel mimicking arteriovenous fistulas or an aneurysm contiguous with dilated uterine arteries in the mid-arterial–capillary phase in 20 patients; it showed contrast brush in the remaining two patients. TAE technical success was achieved in all patients. No major complications were observed in any patients. Fifteen patients were followed up with expectant management after TAE; all but one patient showed no re-bleeding during the follow-up period . One patient showed minor rebleeding, which resolved spontaneously. Seven patients underwent scheduled hysteroscopic resection within 1 week after TAE, and no excessive bleeding was observed during or after the surgical procedure in all seven patients.The characteristic angiographic feature of retained placenta is “dilated vascular channel that mimic low flow AVM.” TAE is a safe and effective treatment to manage retained placenta with abnormal bleeding. Retained placenta is a major cause of postpartum hemorrhage. Notably, retained placenta in the presence of severe postpartum hemorrhage has a reported frequency of 11.4%–33.3% among the patients with severe postpartum hemorrhage  has been established as a treatment option for uncontrollable postpartum hemorrhage  had undergone selective TAE; (b) had a symptomatic vaginal bleeding; (c) clinically diagnosed retained placenta based on the patient’s medical history, clinical examinations, and transvaginal ultrasonographic (TVUS) findings. (d) had no other endometrial diseases. Retained placenta tissue with marked vascularity was confirmed by TVUS in all patients. In addition, biphasic contrast-enhanced (CE) CT (21 patients) or dynamic CE MRI (one patient) was performed to evaluate the location of the retained placenta and its potential feeding arteries.The characteristics of the 22 patients in this study are summarized in Table The decision indication for the procedure was taken by multidisciplinary agreement referring to TVUS and CE-CT or MRI findings. All TAE procedures were performed by experienced interventional radiologists with more than 10 years of experience using digital subtraction angiography equipment . Bilateral internal iliac angiographies with anterior and anterior oblique projections were performed using a 4-Fr or 5-Fr diagnostic catheter with injection of a nonionic iodinated contrast media  at a flow rate of 3–5 mL/sec  through an automatic injector. Then, a 2.7-Fr microcatheter was introduced through the diagnostic catheter into the ipsilateral uterine artery, and selective angiography of the uterine artery was subsequently performed with manual injection of 2–4 mL of contrast media. A 1.6-Fr or 1.9-Fr microcatheter was then advanced distally through the 2.7-Fr microcatheter to the target feeder when uterine angiography showed abnormal findings indicative of retained placenta and/or possible source of bleeding. We adopted triaxial system  to advance the microcatheter into the tortuous uterine artery. In this system it was easier to advance the 1.6-Fr or 1.9-Fr microcatheter because the 2.7-Fr microcatheter stabilized the position and prevented sagging or jumping. If uterine angiography did not show definitive findings of retained placenta, selective catheterization and angiography of the contralateral uterine artery were performed. Furthermore, target embolization was performed using gelatin sponge pieces or a mixture of n-butyl-cyanoacrylate (NBCA) and lipiodol (ratio of 1:3–5) when the 1.6-Fr or 1.9-Fr microcatheter was able to reach the appropriate feeders to the target lesion. If selective catheterization failed or numerous feeders originated from the proximal portion of the uterine artery, empirical embolization was performed with gelatin sponge pieces at the proximal site of the uterine artery. Embolic materials were selected by each operator in accordance with microcatheter reachability and angiographic features including the size and numbers of feeding arteries, as well as the size of the target lesion. After embolization, disappearance of the target lesion was confirmed by selective angiography of the bilateral uterine arteries and the bilateral internal iliac arteries. When residual supply to the target lesion was observed on contralateral angiography, embolization from the feeders of the contralateral uterine artery was performed by the same techniques. TAE technical success was defined as disappearance of the target lesion during the final angiography examination.Two experienced radiologists reviewed all images obtained before TAE treatment in all 22 patients. They evaluated whether the angiographic findings corresponded to retained placenta in each patient. Coronal maximum intensity projection (MIP) images of CE-CT or MRI in the arterial phase were used as a reference to identify retained placenta.The need for each patient to undergo additional hysteroscopic resection after TAE was assessed by the attending gynecologist. Although patients were scheduled for hysteroscopic resection after TAE during the early portion of the study period, conservative management was preferred during the late portion of the study period. Expectant management after TAE was performed in 15 patients (expectant management group); hysteroscopic resection was performed within 1 week after the TAE procedure in the remaining seven patients . Clinical success was defined as the absence of re-bleeding requiring additional treatment in the expectant management group; it was defined as surgical completion without excessive intraoperative bleeding (≥2000 mL) in the surgical management group. Clinical success was investigated by reviewing medical and operative records. In the expectant management group, vascularity of retained placenta on TVUS or CE-CT within 1 week and more than 1 month after the TAE procedure were also evaluated.The angiographic findings and results of selective TAE are summarized in Table Pelvic angiography  showed a dilated vascular channel contiguous with multiple feeders from dilated uterine arteries in the mid-arterial to capillary phase, followed by drainage into the uterine veins in the capillary to venous phase in 20 patients Figs.  and 2. Tn = 6) and large retained placenta with numerous feeders (n = 1).Regarding embolic materials, gelatin sponge (GS) pieces 1–2 mm) were used in 18 patients  and gluing microcatheter in a feeder (n = 1). Transient hypotension resolved with conservative management. In the patient with an NBCA-adhered microcatheter, the amputated catheter tip remained in the left uterine artery.No major complications were observed. Three minor procedure-related complications (CIRSE grade 1) were observed: transient hypotension (n = 8) or disappeared (n = 7). Follow-up TVUS and/or CE-CT at 1 month after TAE showed no abnormal blood flow in the uterus in all 15 patients. Complete hemostasis without recurrent bleeding was achieved in 14 of these 15 patients. In one patient, minimal vaginal bleeding occurred after TAE, but spontaneously disappeared within 1 month.The clinical success rates in the expectant management and surgical management groups were 100% (15/15 patients) and 100% (7/7 patients), respectively. Tables Prevention effect of intraoperative bleeding after TAE are summarized in Table Thus far, few reports have specifically mentioned angiographic features of retained placenta in patients with postpartum hemorrhage . Larger prospective studies are needed to confirm the safety and efficacy of the TAE procedure as a monotherapeutic approach for retained placenta with abnormal bleeding.In summary, the characteristic angiographic feature of retained placenta with vaginal bleeding is a dilated vascular chaneel fed by multiple uterine arterial branches in the arterial to capillary phase, which drains into the uterine vein in the capillary to venous phase. TAE using GS and/or NBCA can be a safe and effective treatment for management of abnormal bleeding caused by retained placenta.


Collagens and their most characteristic structural unit, the triple helix, play many critical roles in living systems which drive interest in preparing mimics of them. However, application of collagen mimetic helices is limited by poor thermal stability, slow rates of folding and poor equilibrium between monomer and trimer. Covalent capture of the self-assembled triple helix can solve these problems while preserving the native three-dimensional structure critical for biological function. Covalent capture takes advantage of strategically placed lysine and glutamate (or aspartate) residues which form stabilizing charge–pair interactions in the supramolecular helix and can subsequently be converted to isopeptide amide bonds under folded, aqueous conditions. While covalent capture is powerful, charge paired residues are frequently found in natural sequences which must be preserved to maintain biological function. Here we describe a minimal protecting group strategy to allow selective covalent capture of specific charge paired residues which leaves other charged residues unaltered. We investigate a series of side chain protecting groups for lysine and glutamate in model peptides for their ability to be deprotected easily and in high yield while maintaining (1) the solubility of the peptides in water, (2) the self-assembly and stability of the triple helix, and (3) the ability to covalently capture unprotected charge pairs. Optimized conditions are then illustrated in peptides derived from Pulmonary Surfactant protein A (SP-A). These covalently captured SP-A triple helices are found to have dramatically improved rates of folding and thermal stability while maintaining unmodified lysine–glutamate pairs in addition to other unmodified chemical functionality. The approach we illustrate allows for the covalent capture of collagen-like triple helices with virtually any sequence, composition or register. This dramatically broadens the utility of the covalent capture approach to the stabilization of biomimetic triple helices and thus also improves the utility of biomimetic collagens generally. A minimal protecting group strategy is developed to allow selective covalent capture of collagen-like triple helices. This allows stabilization of this critical fold while preserving charge–pair interactions critical for biological applications. One of the best known examples is human insulin, a dimer linked by two interchain disulfide bonds.12 beta sheet13–17 and polyproline type 2 secondary18–21 structures have been prepared by this self-assembly followed by covalent capture approach.Inspired by Nature, researchers have utilized covalent capture strategies to stabilize designed self-assembling systems. Alpha helical,20–22 These engineered collagens were found to have improved thermal stability and an increased rate of folding while perfectly preserving the native triple helical fold of the assembled peptides.Our group recently developed a covalent capture technique for collagen triple helices in which isopeptide bonds are formed between the side chains of lysine and aspartate or glutamate.23–26 Covalent capture is particularly important for collagen-like triple helices as they are well known to have very slow rates of folding (hours to weeks) and only moderate thermal stability , two problems that covalent crosslinking can overcome. Our previous work first directed triple helix self-assembly with selective charge paired hydrogen bonding interactions and then covalently linked these amino acids to form isopeptide bonds. This approach only works when the lysine and glutamate residues are in one of two specific sequential geometries with respect to one another forming axial  protecting groups do not adversely impair helix solubility nor (2) disrupt the structure and stability of the triple helix. (3) They must also prevent unwanted isopeptide amide bond formation while (4) not interfering with distal reactivity. Finally, (5) they must be easily removable under relatively mild conditions in good yield. Here we develop methods for selective covalent capture in the presence of multiple charge–pair interactions through the use of a minimal sidechain protection strategy.28,29 Glutamate was protected by OAllyl, OBzl or ODmab. Effective chemical removal of allyl ester from peptides frequently involves the use of palladium catalysts.30–32 Although the development of advanced catalysts can reduce the catalyst's toxicity, the costs remain high and catalyst recovery is problematic. Benzyl esters can be removed by catalytic hydrogenation or saponification.33,34 ODmab protecting group is readily removable with 2% w/v hydrazine, but cyclization side reactions frequently occur.29,35,36 In this work, we have optimized deprotection reaction conditions with the template triple helices for low cost, high yield under aqueous conditions. These methods were then applied to the selective covalent capture of significantly more complex and chemically diverse peptides from SP-A in which (PKGEOG) motifs were added to the termini of the SP-A sequence to facilitate covalent capture . The SP-A derived triple helix has four internal salt-bridges in total and a lysine residue is potentially involved in all the salt bridges. Therefore, protecting the side chain of this specific lysine residue is the most economic strategy. Alternatively, if in a triple helix, there are more positively charged than negatively charged amino acid residues involved in salt bridges, protecting the negatively charged residues would be the most appropriate approach.Two protecting schemes are available: protection of positively or negatively charged amino acid residues. In principle, all charged amino acids could be protected, but this is synthetically more cumbersome and likely would lead to poor aqueous solubility and failure of the helix to fold. Therefore, our goal was to use the minimum number of protecting groups possible. Based on this, either the target lysine(s) or target glutamate(s) were protected, but never both.  capture . SP-A (p22,43,44 Detailed methods are available in the ESI.Peptides were prepared by solid phase synthesis using an FMOC strategy as previously described by us.The model peptide triple helices bearing protecting groups were covalently captured using the carboxylate activating reagents EDC and HOBt. Detailed methods are available in the ESI.2, 25 eq. GSH in 100 mM MES buffer, 37 °C, 30 min,31 we were able to achieve quantitative deprotection of the OAllyl group from monomer peptides, but similar conditions, even with elongated reaction time or higher reaction temperatures, resulted in incomplete deprotection of covalently captured triple helices. The deprotection conditions for Dmab that worked well for monomer peptides caused significant glutarimide/pyroglutamyl side product formation.35,36 We suspect that in the triple helix conformation, the amide proton of the adjacent strand can be close to the glutamate with Dmab protecting group, resulting in a geometry favourable for the cyclization reaction while this scenario happens less frequently in monomer peptides. In contrast to the above difficulties, we found that the simple saponification of benzyl and allyl esters worked surprisingly well. 80 mM K2CO3 at pH 11 was found to deprotect both the OAllyl and OBzl groups with high yield giving a clean mass spectrum of the deprotected trimer.Several deprotection conditions were tested on peptide monomers and covalently captured trimers . In some2CO3 at room temperature, 35 °C and 45 °C with the template triple helices. The reaction progress was followed with UPLC (Fig. S13 and S14The hydrolysis of esters is one of the easiest and most classic reactions but is rarely used in peptide synthesis. We have tested the hydrolysis kinetics of benzyl and allyl esters at different reaction conditions . We firs2CO3 using benzyl ester as an example (2CO3, respectively.We then tested the deprotection at different pH conditions at 45 °C with K example  and S15†2OH also worked successfully.The deprotection of ivDde protecting group from the side chain of lysine residue with 0.5 M NHAfter optimization, we achieved complete deprotection of ivDde, OAllyl and OBzl in covalently captured triple helices or monomeric peptides with conditions 8, 10 and 12, respectively . It shouWe characterized the thermal stability and refolding of the supramolecular and covalently captured triple helices. The melting temperature of the supramolecular KGE triple helices is 46 °C . The refThe selected sequence in the collagenous domain of SP-A has four internal salt–bridge interactions which involves one lysine, one aspartate and two glutamate residues. Depending on the registration, the lysine residue can potentially make contact with each of the negatively charged residues through axial or lateral interactions. Therefore, the minimal side chain protection strategy suggests protection of lysine with ivDde  while leBoth ivDde-protected peptides SP-A1 and SP-A2 folded into triple helices according to the CD results (Fig. S16 and S17The supramolecular SP-A1 and SP-A2 triple helices have a melting temperature of 28 and 35 °C, respectively . The SP-We prepared ccSP-A1 by covalently capturing the N-terminus of the trimer followed by deprotection of lysine. The melting temperature of ccSP-A1 is 35 °C, which is 7 °C higher than its supramolecular counterpart . The refIn this work we demonstrated a minimal protecting strategy for the covalent capture study of collagen triple helices. Our approach allows the covalent stabilization of triple helices with nearly any sequence. This strategy does not appreciably affect collagen triple helix conformation nor solubility. The deprotection conditions studied here avoid the use of organic reagents, instead removing the protecting group easily under aqueous condition. The methods developed here were tested with natural sequences from SP-A which cannot be covalently captured without protecting groups. The covalent triple helix was found to refold more rapidly and have a thermal stabilities 7 °C or 15 °C higher than their supramolecular analogues depending of if a single or double covalent capture strategy was employed. Perhaps most important for applications of triple helices, either approach allows their use in situations where very low concentrations must be used. The selective covalent capture methods described here provide a blueprint for successful stabilization of virtually any collagen triple helix which should expand their use in a large variety of biological applications.The datasets supporting this article have been uploaded as part of the ESI.†Jeffrey D. Hartgerink contributed to the conceptualization of the research. Le Tracy Yu contributed to experimental investigation of the work. Jeffrey D. Hartgerink and Le Tracy Yu both contributed to the experimental designs and writing of the manuscript.There are no conflicts to declare.SC-013-D1SC06361H-s001


Any short RNA that enters the RNA induced silencing complex (RISC) can kill cells through miRNA-like RNA interference when its 6mer seed sequence (pos. 2–7 of the guide strand) has a G-rich nucleotide composition. G-rich seeds mediate 6mer Seed Toxicity by targeting C-rich seed matches in the 3′ UTR of genes critical for cell survival. The resulting Death Induced by Survival gene Elimination (DISE) predominantly affects cancer cells but may contribute to cell death in other disease contexts. This review summarizes recent findings on the role of DISE/6mer Seed Tox in cancer; its therapeutic potential; its contribution to therapy resistance; its selectivity, and why normal cells are protected. In addition, we explore the connection between 6mer Seed Toxicity and aging in relation to cancer and certain neurodegenerative diseases. In this review, we will summarize what is currently understood about a novel mechanism that regulates cell fate, 6mer Seed Toxicity. We posit that 6mer Seed Tox evolved to be a powerful anti-cancer mechanism with potential relevance to the etiology of other diseases. We usually refer to cancer as the disease that patients present with in the clinic. However, cancer begins at the single cell level. A single cell may accumulate mutations and become neoplastically transformed. The acquisition of mutations and subsequent genomic instability is one of the central hallmarks of cancer . Cancer RNAi is mediated by micro (mi)RNAs, small (18-22 nt long) noncoding RNAs that are generated from long double-stranded (ds)RNAs 15). miRNAs are transcribed in the nucleus as hairpins that are processed by the Drosha/DGCR8 microprocessor , and tra. miRNAs Both miRNAs and RNAi pathway genes are highly conserved throughout evolution. The human genome codes for more than 2300 miRNAs  and someIn 2014, we reported that a number of siRNAs and shRNAs designed to silence either the death receptor CD95/Fas or its ligand, CD95L, killed all tested cancer cells by activating multiple cell death pathways. No pharmacological inhibitor or knockdown of any single gene was found to block this form of cell death. Repeat treatment with sh/siRNAs was effective in killing, suggesting that cancer cells could not easily develop resistance . Subsequ6merdb.org). The screen revealed that the rules of toxicity were very similar in all cell lines suggesting that this kind of toxicity kills cancer cells regardless of the tissue or species of origin -5p (the miR-34-5p family) is a master tumor suppressive miRNA family that was found to exert its tumor suppressive function, at least in part, through 6mer Seed Tox . Its 6meA duplex . These dA duplex ). miR-34A duplex . While nHowever, not all tumor suppressive miRNAs engage in 6mer Seed Tox. Major miRNA families such as let-7 and miR-200 do not carry toxic 6mer seeds, and instead exert their tumor suppressive activity by inducing and/or maintaining cell differentiation –49, or iThe identification of endogenous tumor suppressive miRNAs that are toxic to cells through 6mer Seed Tox suggests that certain miRNAs are purposefully expressed to target essential/survival genes Fig. B. Their We observed in sequence logos of miRNAs grouped by age that most younger miRNAs (< 10 million years old) contain Gs as the most abundant nucleotide in five out of the six positions of their 6mer seed . These iBy ranking all mature miRNAs according to their predicted 6mer Seed Toxicity we could efficiently separate oncogenic miRNAs from tumor suppressive ones .The existence of specialized miRNAs that contain G-rich 6mer seeds raised the question of how toxic miRNAs were selected during evolution, and how to prevent accidental cell death. By analyzing all major ~ 2600 mature human miRNAs we found two important features of miRNAs that fundamentally support the concept that DISE is an evolutionarily conserved process :We obserDespite the observed selection for more non-toxic miRNAs over time, toxic miRNAs with G-rich seeds have been fixed in the genome. Our analysis revealed that some of the best characterized tumor suppressive miRNAs carry a toxic seed . They arOur data suggest that the 6mer Seed Tox concept is not limited to miRNAs. Any short (s)RNA that can enter the RISC and contains a G-rich seed could have anti-cancer activity. We demonstrated this in HCT116 Drosha knock-out (KO) cells which are devoid of most endogenous miRNAs. When we pulled down Ago proteins 1–4 in wild type (wt) and Drosha KO HCT116 cells and sequenced the bound RNAs, as expected, we observed that the RISC content of wild-type HCT116 cells consisted primarily of miRNAs  . In cont6merdb.org). Interestingly, vtRNA2–1 is one of the predominant RNA species in the RISC of Drosha KO cells [A recent study provided evidence that a small noncoding RNA can exert tumor suppressive activities through RNAi. vtRNA2–1/nc886 is a non-coding vault RNA (vtRNA) that is cleaved by Dicer to give rise to a dsRNA. Its 3p arm is predominantly expressed and carries the seed GCGGGU, a 6mer seed sequence that was toxic to all six human and murine cancer cell lines tested in our 6mer seed screen  two CD95L-derived DISE-inducing siRNAs coupled to HDL mimetic nanoparticles . Repeate6merdb.org). This miRNA is therefore a good candidate for reducing tumor growth through 6mer Seed Tox. Similar to our studies, these groups did not report any general toxicity in the treated mice.A number of published studies demonstrate the efficacy and safety of employing RNAi to treat cancer, and their data are consistent with our model of 6mer Seed Tox. For instance, i.p. delivery of the tumor suppressive miR-193a-3p in liposomes substantially reduced the growth of HCT116, HT29, and MDA-MB-231 tumors when grown subcutaneously in xenografted mice . InteresWe demonstrated that a large part of the antitumor activity of miR-34a-5p stems from its 6mer Seed . InteresNormal tissues may be less susceptible to toxic siRNAs than cancer cells. This notion is supported by a number of observations: 1) Treating OvCa bearing mice with either DISE-inducing siRNAs or CAG TNR-based siRNAs did not cause any toxicity in the treated mice , 64; 2) The miRNA protection model. Drosha and Dicer KO cells are hypersensitive to various DISE-inducing stimuli [ stimuli , 44, 58. stimuli ; 2) Dros stimuli , 44, 58.While there could be different explanations for the cancer selectivity of the toxic siRNAs, there are a number of not mutually exclusive mechanisms at play involving miRNA/RNAi pathway components that could influence the selectivity. Hence, we are proposing four different models:2)The differential Ago stability model. Dicer or DGCR8 KO mice have demonstrated that in normal cells, such as embryonic stem cells or mouse embryonic fibroblasts, Ago proteins are degraded in the absence of miRNAs . Inussed in , 30. Theussed in . Thus, t3)The genomic stability protection model. Almost all genes involved in miRNA biogenesis, Drosha, DGCR8, Dicer, Exportin-5 (XPO5), and Ago2 have been found to be involved in the DNA damage response and/or DNA repair [A repair , 71–77: A repair ; both DiA repair , which fA repair ). In addA repair . BecauseA repair , the senA repair .4)The survival gene dependence model. It is conceivable that cancer cells, which in general proliferate at a higher rate than mostly quiescent normal cells, express survival genes at higher levels and hence are more dependent on their activities for cell survival than normal cells.There is evidence that highly expressed miRNAs with nontoxic 6mer seeds protect normal cells from DISE induction. In most cells, more than 95% of all RISC-bound RNAs are miRNAs, with most miRNAs carrying nontoxic seeds . In contRegardless of the mechanism underlying the cancer selectivity of DISE, there is early evidence of a therapeutic window. By activating DISE in the correct context, one could kill cancer cells and spare normal cells. Further studies will seek to understand and extend this therapeutic window.Despite recent therapeutic advances, recurrent ovarian cancer—particularly the most prevalent and most malignant form, high-grade serous ovarian cancer (HGSOC)—is incurable and has a poor prognosis, with a median survival of 40.7 months . The maimiRNAs have been linked to cancer therapy resistance in countless reports in almost all cancers . For ovaWe previously showed that treating cancer cells with chemotherapeutic drugs such as etoposide, carboplatin and doxorubicin caused upregulation of miRNAs with toxic 6mer seeds . Indeed,miRNAs in cancer are almost uniformly quantified using total RNA. However, our data suggest that, instead, it is more meaningful to analyze RISC-bound sRNAs given the fact that most mature miRNAs are not Ago associated  and thatTo provide a standardized tool to analyze 6mer Seed Tox, we recently developed SPOROS (Greek for seed), an automated bioinformatics pipeline for the analysis of small RNA Seq data . SPOROS While cancer cells can hardly become completely resistant to 6mer Seed Tox, they could however increase their tolerance through two possible mechanisms: 1) by altering the ratio of toxic-to-nontoxic sRNAs towards more nontoxic ones and 2) by acquiring mutations in the targeted 6mer seed matches. When we determined that G-rich seeds were the most toxic in the screen of the 4096 6mer seeds, we were surprised to note that C-rich seeds were much less toxic . From a Single nucleotide polymorphisms (SNPs) represent another example of mutational changes that may affect disease outcome. SNPs are found in miRNAs and can occur in the miRNA seed , 90. An In addition to these potential mechanisms affecting the activity of 6mer Seed Tox, another mechanism could involve the generation of toxic miRNAs, as this process must be tightly regulated at multiple levels. This could occur through RNA modifications or the changes in the expression or localization of RNA binding proteins. Dysregulation of these pathways in certain conditions such as advancing age could reduce the potency of the 6mer Seed Tox anti-cancer mechanism.It is clear that cancer incidence increases with age. Multiple mechanisms have been described to contribute to this phenomenon . One estAs noted above, the loss of cytosines in the genome may hinder the anti-cancer function of 6mer Seed Tox. As cytosines in the 3′ UTR of survival genes are lost, this could decrease the probability that these genes are targeted by toxic G-rich sRNAs directing the RISC. Over time, the accumulation of C > T mutations could gradually relieve cells from 6mer seed Tox surveillance, resulting in the development of cancer. Thus, the accumulation, specifically of C > T mutations, provides a link between increased cancer incidence and old age through 6 mon Seed Tox.Assuming that normal cells are largely protected from 6mer Seed Tox by high concentrations of miRNAs with nontoxic seeds, this raises the question of what happens if a tissue loses this protection Fig. B. We hypDrosophila impairing miRNA processing dramatically enhanced neurodegeneration caused by the CAG repeat gene ATXN3 [There is ample literature to support the notion that miRNAs are neuroprotective (reviewed in ). In facne ATXN3 . Multiplne ATXN3 , 100). Mne ATXN3 . Interesne ATXN3 , suggestIn fact, it has been shown that increasing miRNA levels can be neuroprotective. Enoxacin is an antibiotic drug that stabilizes Dicer/TRBP resulting in a global upregulation of miRNAs –105. In In summary, miRNA (or miRNA biogenesis enzyme) levels may drop in the aging brain resulting in a progressive loss of protective miRNAs. This in turn could lead to a relative increase in toxic sRNAs in the RISC  exhibit signs of general toxicity, or whether other mechanisms underlie sensitivity. If miRNA downregulation underlies sensitivity, 6mer Seed Tox could be relevant in additional disease contexts. For example, the tissues of the spleen and adipose tissue have also been shown to lose expression of Dicer and/or Exportin-5 with age, and thus may also have globally downregulated miRNA levels . Therefo


The effects of corrugated grain boundaries on the frictional properties of extended planar graphitic contacts incorporating a polycrystalline surface are investigated via molecular dynamics simulations. The kinetic friction is found to be dominated by shear induced buckling and unbuckling of corrugated grain boundary dislocations, leading to a nonmonotonic behavior of the friction with normal load and temperature. The underlying mechanism involves two effects, where an increase of dislocation buckling probability competes with a decrease of the dissipated energy per buckling event. These effects are well captured by a phenomenological two-state model, that allows for characterizing the tribological properties of any large-scale polycrystalline layered interface, while circumventing the need for demanding atomistic simulations. The resulting negative differential friction coefficients obtained in the high-load regime can reduce the expected linear scaling of grain-boundary friction with surface area and restore structural superlubricity at increasing length-scales. Achieving ultra-low friction at macroscopic scales is highly desirable. In this work molecular dynamics simulations of graphitic contacts incorporating corrugated grain boundaries reveal an unusual non-monotonic variation of friction with normal load and temperature due to dynamic buckling effects. Recent studies have reported structural superlubricity in nano- and micro-sized monocrystalline samples of layered materials, where misoriented lattices7 or mismatched lattice constants13 facilitate the necessary incommensurability. However, scaling up structural superlubricity towards macroscopic dimensions inevitably involves forming junctions between surfaces of polycrystalline nature.Reduction of energy dissipation and wear is of critical importance and growing demand in a wide range of areas, including mechanical, electronic, and biological systems. Compared to traditional lubrication schemes, structural superlubricity, featured as extremely low friction due to the effective cancellation of lateral forces at incommensurate crystalline interfaces, has emerged as a novel route toward efficient reduction of friction and wear14. Since the friction between misaligned graphitic patches is known to be negligibly small3, one could expect that superlubricity would prevail also in large-scale PolyGr interfaces, pending that the patch dimensions remain at the nanoscale. However, PolyGr GBs often exhibit out-of-plane corrugation17, which may introduce substantial friction20 and enhance wear21. To control and eliminate such undesired effects one must fully understand the mechanisms underlying energy dissipation at elongated graphene GBs. This, in turn, requires elucidating collective effects between different topological defects along the GBs under persistent shear.The simplest example of polycrystalline monoatomic two-dimensional (2D) surface is polycrystalline graphene (PolyGr), which is composed of randomly shaped and oriented single crystalline graphene patches separated by sharp grain boundaries (GBs). The latter are characterized by chains of lattice dislocations often including pentagon–heptagon pairs27.Here, we reveal that collective dynamic effects at PolyGr GBs may lead to unusual nonmonotonic variation of the friction with normal load and temperature. Notably, at room temperature or above we find a monotonic decrease of friction with the external normal load for the systems considered, resulting in negative differential friction coefficients. The discovered phenomenon is of general nature and is expected to occur in other polycrystalline layered materials that demonstrate out-of-plane GB deformations. Moreover, the knowledge gained in this study may provide insights regarding universal mechanisms of energy dissipation appearing in extended multi-contact rough interfaces, where the formation and rupture of contacts dictates the frictionθ0 = 38.2°, and a substrate consisting of a layer of PolyGr including two patches with orientation angles of θ1 = 0°  and the armchair direction of the lattice of the relevant layer. The PolyGr layer contains two GBs composed of lines of separated pentagon–heptagon pair dislocations along the GB (y-axis) direction  reaches ~1.4 Å, consistent with previous experimental measurements16. After annealing the entire six layers structure at 1000 K and zero normal load, most of the dislocations exhibit considerable out-of-plane deformations, protruding upward or downward as shown in the lower panel of Fig. Our model system for studying the friction over extended graphene GBs is shown in Fig. ion Fig. . The disGPa Fig. .Fig. 1Mox direction  and the PolyGr layer (l4). The generated heat is removed from both slider and substrate without affecting the dynamics at the sheared interface. See “Methods” and Supplementary Methods for further details regarding the simulation model and protocols.In the sliding simulations, normal loads of up to ~2.3 GPa are applied. The top layer is kept rigid and is shifted with a constant velocity of ion Fig. . Since t−4 and −8.1 × 10−5, respectively, well within the superlubric regime. When the temperature is increased to 50 K the overall friction increases and the maximal friction point shifts towards lower normal loads. When the temperature is further increased to 150 K the maximum completely disappears and the friction reduces monotonically with the normal load exhibiting negative differential friction coefficients across the entire load range considered28. At 300 K a similar behavior is observed with overall reduced friction. The differential friction coefficients calculated in this case are between −1 × 10−4 and −8.9 × 10−6. Compared to experiments, the maximal frictional stress obtained in our simulations (~160 kPa) is less than one order magnitude higher than that measured for misaligned homogeneous pristine graphitic contacts29, and comparable to that of aligned graphite/hexagonal boron nitride (h-BN) heterojunction10. See Supplementary Note The dependence of the interfacial friction between the PrisGr and PolyGr surfaces on the normal load and temperature are presented in Fig. T = 150 K. As the normal load increases, the overall friction force decreases, and the position of maximal friction shifts to lower temperatures.The friction–temperature relation, shown in Fig. T = 0 K. Analyzing the energy dissipation routes by calculating the steady-state dissipation power through the damped layers in different directions  equilibrium molecular dynamic simulations of the six-layered model system presented in Fig. z coordinate of the atom with largest RMS corrugation in the dislocation region. A typical example of such an atomic trajectory is presented in the inset of Fig. The dynamic snap-through buckling phenomenon indicates that the GB dislocations exhibit bi-stable behavior characterized by the upward and downward protruding states separated by transition energy barriers (TEBs). An estimate of the corresponding barrier heights can be obtained by considering the thermally activated dislocation buckling process in the absence of sliding. To this end, we performed room temperature (T = 0 K) the snap-through bi-stability can be triggered by the sliding process via two main effects: (i) variation of the snap-through barrier along the scan-line and (ii) shear-force induced out-of-plane motion of atoms in the dislocation region. The former results from the registry dependence of the vertical buckling energy profile, as discussed above, whereas the latter, which may be caused by local heating, is found to be of minor importance under our simulation conditions, as demonstrated below by comparison to a phenomenological two-state model. At zero normal load, only a few shear-induced buckling events occur  and in the absence of normal load, thermal fluctuations can assist overcoming the TEBs for buckling during sliding, activating more dislocations to buckle and increasing their snap-through frequency . The results demonstrate similar dynamic snap-through buckling of GB dislocations and a corresponding nonmonotonic load dependence of the friction  the distribution of TEBs corresponding to different dislocations is uniform within a given range  thnge Fig. ; and  to the overall friction is small, as their tribological properties resemble those of a pristine interface. Therefore, the phenomenological model developed herein can serve as an efficient tool to characterize the frictional properties of complex interfaces consisting of involved GB geometries, as long as friction is dominated by buckling events. This may help reducing the dependence on explicit demanding atomistic simulations of large-scale polycrystalline mosaic interfaces and focusing computational efforts on understanding specific microscopic mechanisms of interest.Aiming to scale-up structural superlubricity in layered material interfaces implies the inevitable appearance of GBs at the sliding interface. As shown above, the scaling of their dissipative contribution with contact area is stronger than the sublinear scaling found for pristine incommensurate layered contacts36, which provides energetically favorable GBs with dislocation content and structural properties in excellent agreement with experimentally measured GBs35, and introduces minor strain effect (see Supplementary Methods for details). Periodic boundary conditions are applied for all six layers along both lateral directions, thus mimicking an infinite interface . The intralayer and interlayer interactions are modeled with the second-generation reactive empirical bond order (REBO) potential37 and the registry-dependent ILP42 with the refined parameters42 that provide accurate results up to high external pressures43, respectively. This combination was extensively investigated in recent years and found to provide very good agreement with experimental results of the compressibility43, thermal conductivity44, tribological properties10, and phonon dispersion43 of layered materials interfaces. Furthermore, this approach yields GB corrugations, topographies, and energies comparable to available experimental results and DFT calculations18. All simulations were performed using the LAMMPS package45.The structure of the PolyGr layer is generated using a Voronoi tessellation method developed by Shekhawat and colleagues−1 was applied to the relative velocities (with respect to the velocity of the rigid top slider layer) of each atom in the second layer from top (slider) and to the velocities of each atom in the fifth layer from top (substrate). For finite temperature simulations, Langevin thermostats (rather than mere viscous damping) are employed to these layers. This mimics the energy dissipation channels  through both slider and substrate in experiments, with minor effect on the dynamics of the two layers at the shear plane (see Supplementary Note −1 is also applied to the vertical velocities of the atoms in the top slider layer46. See Supplementary Methods for further details regarding the simulation model and protocol.To remove the heat accumulated during sliding, velocity damping with a damping coefficient of 1.0 psSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6