Hearing loss (HL) is an extremely common neurosensory deficit with a heterogeneous etiology including environmental and genetic causes. The incidence of profound sensorineural HL in the United States is 186 per 100,000 births (Morton and Nance http://hereditaryhearingloss.org/). In view of our knowledge that a large number of genes are involved with HL, and that mutations in each gene would account for a small proportion of all HL, the discovery that mutations in a single gene GJB2 (DFNB1 locus), encoding connexin 26 (Cx26), accounted for a large proportion of recessive NSHL, came as an unexpected surprise . Interestingly, del Castillo et al. , cell‐cell junctions (includes gap‐junction proteins such as connexin‐26 and connexin‐30 and others), membrane transporters such as SLC26A4 encoding the pendrin protein and the other potassium channels (KCNQ4) as well as regulatory elements including several transcription factors. The discovery of various genes, mutations in which can cause Usher syndrome, have helped elucidate the molecular mechanisms underlying hair bundle development and mechano‐electrical transduction . The advent of newborn hearing screening has made it possible to detect HL by 2–3 months of age, and infants who begin rehabilitation by 6 months of age have much improved language and scholastic outcomes compared to those whose HL is detected after 6 months of age  are now offered to infants and placed in both ears to offer the maximal benefit. However, management approaches and options for sensorineural hearing loss continue to lag behind the significant understanding of the basic genetic defects causing HL. The past few years have seen a glimmer of hope for treatments focused on the basic genetic defect for HL, albeit in animal models. Hearing restoration to near normal was demonstrated in a gene therapy study which reinstated the expression of vesicular glutamate transporter 3 (VGLUT3) using postnatal AAV‐mediated delivery (Akil et al. This is an exciting time for those involved in the field of hearing loss, given the explosion in our understanding of the molecular basis of deafness, and the mechanisms and function leading to hearing loss. The implementation of audiologic newborn screening allows early diagnosis of HL with appropriate interventions to prevent language delay. However, as we have got a glimpse into the future of specific gene‐ and mutation‐based therapeutics for HL, it becomes imperative that we focus our attention on enhancing the EHDI programs with complementary molecular diagnosis in newborns for an etiologic diagnosis. Until we determine a specific etiologic diagnosis for the HL, offering timely gene‐based therapeutics may not be feasible. In contrast, it is possible that modalities for hair cell regeneration may not need a specific etiologic diagnosis. However, based on experience with newborn metabolic screening, we have learnt that understanding the natural history and having a specific diagnosis are crucial to implementing state of the art patient care and therapies. There is also a need for additional research to understand the molecular basis for the auditory neuropathy spectrum disorders as well as age‐related hearing loss in the future.


Visual hallucinations that arise following vision loss stem from aberrant functional activity in visual cortices and an imbalance of activity across associated cortical and subcortical networks subsequent to visual pathway damage. We sought to determine if structural changes in white matter connectivity play a role in cases of chronic visual hallucinations associated with visual cortical damage.DTI) and probabilistic fiber tractography to assess white matter connectivity in a patient suffering from continuous and disruptive phosphene (simple) visual hallucinations for more than 2 years following right occipital stroke. We compared these data to that of healthy age‐matched controls.We performed diffusion tensor imaging  of white matter tracts in the patient connecting the visual cortex with frontal and temporal regions, and also in interhemispheric connectivity between visual cortices.fMRI) data showing imbalanced functional activity of the same regions in this patient . Long‐term adaptive and disruptive changes in white matter connectivity may account for the rare nature of cases presenting with chronic and continuous visual hallucinations.Cortical remapping and the disruption of communication between visual cortices and remote regions are consistent with our previous functional magnetic resonance imaging ( The type of hallucinatory image is related to the site of damage along the visual pathway, and/or the visual processing function of the implicated region(s). Lesions of the optic radiations, calcarine, and early visual cortices can result in simple (phosphene) hallucinations, whereas involvement of higher visual processing areas is associated with complex visual hallucinations ffytche, .Previous functional magnetic resonance imaging (fMRI) studies have shown visual hallucinations following visual pathway damage are associated with disorganized functional activity in visual cortices across both hemispheres, and in interconnected cortical and subcortical networks  to the lesion site to provide relief from hallucinations that were interfering with quality of life. fMRI showed aberrant functional activity in visual cortices, and an imbalance of activity across frontal, parietal, temporal, and cerebellar regions prior to rTMS treatment  with no history of neurological disorders, and normal or corrected‐to‐normal vision (>0.2 logMAR) were recruited. All participants were right‐handed with no known contraindications to MRI. All participants had taken part in a previous fMRI study . Whole‐brain diffusion‐weighted scanning, T1‐weighted, and T2‐weighted turbo spin echo fluid‐attenuated inversion recovery (FLAIR) imaging sequences were obtained. Full details on MRI acquisition are provided in Supporting Information.Imaging was acquired with a 3‐Tesla Siemens Magnetom2.3http://www.fmrib.ox.ac.uk/fsl).All DTI analyses were performed using FSL’s DTI processing pipeline . Full details on image processing and normalization to standard space are provided in Supporting Information.2.3.2t test for single case studies  White‐Matter Tractography Atlas , consistent with the location of the patient’s lesion site. Tracts of interest were generated to cortical regions of interest based on our previous fMRI findings, which showed functional differences in blood oxygen level‐dependent (BOLD) signal between the patient  and controls  using this method. The same applies to interhemispheric visual cortex tracts.For ease of comparison of probabilistic fiber tractography between the patient and controls, we only present data of one control example here. The results of probabilistic tractography for all other controls are presented in Supporting Information Figures No differences were observed between the patient and controls for intrahemispheric tracts seeded from the visual cortex to superior and middle frontal gyri, and postcentral gyrus. Probabilistic tractography did not reveal tracts from the visual cortex to the middle temporal gyrus or parietal lobules in the patient or controls.4Diffusion imaging highlights changes in diffusion indices of major white matter tracts associated with the patient’s lesion site compared with controls. These differences are consistent with regions previously implicated in visual hallucinations. Moreover, probabilistic fiber tractography demonstrates changes in white matter tracts between the visual cortex and remote regions in the patient that are consistent with previously observed alterations in functional activation between the patient and controls.4.1In the patient, we observed incomplete intrahemispheric tracts from the ipsilesional visual cortex to superior temporal and middle temporo‐occipital gyri Figure . AlthougChanges in FA and/or AD of the superior  and inferior longitudinal fasciculi in the patient Table  are therAlterations in interhemispheric white matter connectivity between visual cortices in the patient Figure  offer fu4.2The marked displacement of the ipsilesional optic radiations in the patient Figure  suggestsLower FA and increased RD were observed in the contralesional corticospinal tract of the patient Table . An incrChanges in white matter tracts in the patient at both ipsi‐ and contralesional hemispheres stem from altered intra‐ and interhemispheric communication, for example, major forceps, or commissural tracts. Early lesions studies of the occipital lobe in animals show retinotopic reorganization and changes of neuronal response properties in the ipsilesional hemisphere, and aberrant distribution of callosal connections in both ipsi‐ and contralesional hemispheres , some differences in diffusion tensor indices between the patient and controls  and cortical remapping as the root of disorganized functional activity in the patient that leads to the continued perception of visual hallucinations. Cortical reorganization may offer explanation for the chronic and persistent nature of the hallucinations in this patient, differentiating this case from the more common reports of acute visual hallucinations (Manford & Andermann, We would like to thank all participants for their contribution.None declared. Click here for additional data file.


SCI) is a significant health burden worldwide which causes permanent neurological deficits, and there are approximately 17,000 new cases each year. However, there are no effective and current treatments that lead to functional recovery because of the limited understanding of the pathogenic mechanism of SCI. In recent years, the biological roles of long non‐coding RNAs (lncRNAs) in SCI have attracted great attention from the researchers all over the world, and an increasing number of studies have investigated the regulatory roles of lncRNAs in SCI. In this review, we summarized the biogenesis, classification and function of lncRNAs and focused on the investigations on the roles of lncRNAs involved in the pathogenic processes of SCI, including neuronal loss, astrocyte proliferation and activation, demyelination, microglia activation, inflammatory reaction and angiogenesis. This review will help understand the molecular mechanisms of SCI and facilitate the potential use of lncRNAs as diagnostic markers and therapeutic targets for SCI treatment.Spinal cord injury ( Spinal cord injury (SCI) is a severe trauma often caused by car accidents or falls, and it is characteristic of high incidence, high morbidity and huge cost via mediating chromatin, transcriptional and post‐transcriptional modification, and lncRNAs have been regarded as a new frontier in the study of many human diseases Long non‐coding RNAs (lncRNAs) are defined as thousands of RNA transcripts longer than 200 nucleotides in length with no protein‐coding potential, and lncRNAs have attracted great attention from the researchers all over the world In this review, we overviewed the recent studies on the molecular functions of lncRNAs involving in spinal cord injury, and further discussed their potential roles of diagnosis and prognostic biomarkers and therapeutic targets, and this review aimed to help deepen the current understanding of lncRNAs in spinal cord injury.Non‐coding RNAs (ncRNAs) cover more than 98% of the human genome i) sense lncRNAs, lncRNAs overlap with one or more exons from another transcript in the same strand; (ii) antisense lncRNAs, lncRNAs overlap with one or more exons from another transcript in the opposite strand; (iii) bidirectional lncRNAs, lncRNAs that share their promoter with another gene in the opposite strand and are initiated <1000 base pairs away in close genomic proximity; (iv) intronic lncRNAs, lncRNAs initiated completely within an intron of a protein‐coding gene without overlapping exons; and (v) intergenic lncRNAs , those that are independent transcripts located between two genes According to their location in the genome, lncRNAs can be classified into five categories: (i) scaffold molecules for providing stabilization in chromatin modification complexes; (ii) providing molecular guides for localization of their binding targets; (iii) as signals, regulated the expression of target genes by recognizing the transcription factors; (iv) as decoys, binding to molecules and block their role on their target genes; (v) acting as miRNA ‘sponges’ to regulate mRNA activity by sharing common miRNA‐binding sites with mRNAs; and (vi) acting as enhancer RNAs or even encoding short peptides with regulating function The main functions of lncRNAs are as follows:  ≤0.001 and a fold change ≥2 were considered as differentially expressed et al. demonstrated that seven lncRNAs that were detected in the adult rat spinal cord showed significant expressional changes, and among the seven lncRNAs, two  were up‐regulated, one (RGD1559747) was down‐regulated, four  were up‐regulated at 1 day post‐SCI, and then subsequently down‐regulated at 4 and 7 days post‐SCI et al. used a microarray method in a contusion SCI mouse model at 1 day, 3 days, 1 weeks and 7 weeks following SCI, and the results showed that few changes were found at 1 day following SCI and the changes peaked 1 week following SCI and subsequently decreased compared with sham operation group After SCI, a series of pathophysiological events occur at molecular and cellular levels. To understand the changes in the expression levels and the corresponding regulatory functions of lncRNAs following SCI, previous studies have performed microarray method and RNA sequencing and found that a large number of lncRNAs are found in the spinal cord and changed following SCI After SCI, permanent neuronal loss is a major obstacle and will result in functional disfigurement, so enhancing the survival of neurons is critical for recovery in patients with SCI in vitro and might play a detrimental role in the pathophysiology of SCI via up‐regulating the expression of inflammatory cytokine tumour necrosis factor α (TNF‐α), and the up‐regulation of TNF‐α was possible via competitively binding miR‐466 l In the central nervous system, astrocytes are the most abundant glial cell type via increased remyelination‐inhibitory molecules et al. established dynamic expression profiles of lncRNAs at different development stages of oligodendrocyte, and they found that overexpression of lncOL1 promotes the differentiation of precocious oligodendrocyte in the developing brain and inactivation of lncOL1 could cause the defects in CNS myelination and remyelination after injury via interaction with Suz12 Myelin sheath, the insulating layer surrounding the axon, is necessary for maintaining structural and functional integrity of neural circuits in vertebrate spinal cords via the Toll‐like receptor signalling pathway in early brain injury (EBI) following subarachnoid haemorrhage (SAH) et al. demonstrated that lncRNA SNHG14 could increase the expression of PLA2G4A by inhibition of miR‐145‐5p, which resulted in the activation of microglia Inflammation is a crucial biological process in response to injury, infection and trauma suffered by cells or tissues, and it involves the cells present within the central nervous system (CNS), including the neurons, macroglia and microglia et al. suggested that long intergenic non‐coding RNAs could be novel drivers of human lymphocyte differentiation via activating the AHRR‐NF‐κB pathway After SCI, the inflammatory response involves the activation of microglia and the infiltration of neutrophils, monocytes and lymphocytes via facilitating the up‐regulation of HIF‐1α by sponging to miR‐153‐3p via inhibition of notch signalling After SCI, injury to vessels may lead to haemorrhage SCI is a complex process integrating multiple related targets and signalling pathways in the nervous, immune and vascular systems, accompanied by many cellular and molecular mechanisms; however, the molecular mechanisms of SCI are not yet completely understood. Furthermore, no effective therapy is currently available to control the secondary injury following SCI Moreover, several issues will be the research focus for further studies. First, the majority of the studies on the effect of lncRNAs on SCI are preclinical animal studies, and further investigations applying to humans will be needed in order to improve the transition to the clinic. Second, an appropriate delivery system is required for implementation of lncRNA therapy, so how to choose an appropriate vector which ensures the successful delivery of the lncRNAs to the desired targets is the future research direction. Furthermore, most of molecular mechanisms of lncRNAs in SCI are still unclear, so further investigations a deeper understanding of the causes and consequences of the dysregulation of lncRNAs in SCI and choosing the most promising lncRNAs will push the future development of lncRNAs for therapeutic applications, including the treatment of SCI.The authors have no conflict of interests to disclose.


Ex vivo‐expanded stem cells have long been a cornerstone of biotherapeutics and have attracted increasing attention for treating intractable diseases and improving tissue regeneration. However, using exogenous cellular materials to develop restorative treatments for large numbers of patients has become a major concern for both economic and safety reasons. Advances in cell biological research over the past two decades have expanded the potential for using endogenous stem cells during wound healing processes, and in particular, recent insight into stem cell movement and homing has prompted regenerative research and therapy based on recruiting endogenous cells. Inspired by the natural healing process, artificial administration of specific chemokines as signals systemically or at the injury site, typically using biomaterials as vehicles, is a state‐of‐the‐art strategy that potentiates stem cell homing and recreates an anti‐inflammatory and immunomodulatory microenvironment to enhance in situ tissue regeneration. However, pharmacologically coaxing endogenous stem cells to act as therapeutics in the field of biomedicine remains in the early stages; its efficacy is limited by the lack of innovative methodologies for chemokine presentation and release. This review describes how to direct the homing of endogenous stem cells via the administration of specific signals, with a particular emphasis on targeted signalling molecules that regulate this homing process, to enhance in situ tissue regeneration. We also provide an outlook on and critical considerations for future investigations to enhance stem cell recruitment and harness the reparative potential of these recruited cells as a clinically relevant cell therapy. We also critically evaluate their roles in biomaterials‐based stem cell homing and accommodation.Similar to strategies applied to improve the homing and engraftment of exogenously transplanted cellular materials in recipient tissues e.g. see , 27, 28,in vitro cell expansion procedures, several other technical hurdles must be addressed before the clinical utility of such stem cell therapies for combating human diseases can be realized e.g. see in situ tissue regeneration e.g. cell‐matrix and cell‐cell contacts), several cell‐free tissue‐engineering approaches have been developed based on biomaterials and the local or systemic administration of biologics or small molecules that may reverse the impaired regenerative microenvironment due to disease and/or induce homing of circulating stem cells for regeneration e.g. the bone marrow) are activated in the circulation by extracellular signals and reach the injury site with the aid of blood flow. These stem cells can be arrested within the local vasculature in response to injury and transmigrate across the endothelium to replenish the local niche and/or participate in regeneration ‐1α, stem cell factor (SCF), granulocyte colony‐stimulating factor (G‐CSF) and monocyte chemotactic proteins (MCPs) via local and systemic actions et al. in situ vascular regeneration, the controlled administration of substance P from the electrospun membranes of a vascular graft generated by mixing substance P‐bound poly (l‐lactide‐co‐ε‐caprolactone) (PLCL) and linear PLCL in appropriate proportions enhanced the recruitment of human BMMSCs, leading to the regeneration of abundant blood vessels in the explants in situ cell differentiation during the second stage; both features are key to ensuring effective bone regeneration in situ vascular regeneration; substance P was bound to recruit endogenous reparative cells, while heparin was conjugated to suppress thrombogenic responses by inducing microphages (Mφs) to polarize into the M2 phenotype in vitro propagation while sustaining the active state of MSCs in vitro expansion and manufacture of cellular materials.When sensory nerves are injured, peripheral sensory neurons release substance P, which is an 11‐amino‐acid peptide and a nociceptive factor that functions as a neuromodulator and neurotransmitter and plays a role in reparative neovascularization in vivo physiological conditions, quiescent haematopoietic stem cells (HSCs) or haematopoietic progenitor cells are maintained in bone marrow stroma through chemokine signalling between the chemotactic factor SDF‐1α, also termed chemokine (C‐X‐C motif) ligand 12 (CXCL12), and the G protein‐coupled receptor C‐X‐C chemokine receptor 4 (CXCR4) et al. N‐isopropylacrylamide) (PNIPAAm) gates grafted on its outer pore surfaces that provided a swollen‐shrunken property in response to temperature changes de novo pulp regeneration in pulpectomized mature teeth in a canine model via a radially oriented collagen scaffold Under in situ regeneration of a specific target, SDF‐1 is commonly combined with other therapeutic agents, and cooperative effects continue to be identified; some examples include the use of SDF‐1 in combination with TGF‐β1 and BMP‐2 for site‐targeted cell trafficking and tissue‐specific differentiation via physical adsorption, an initial quick release of the adsorbed homing agent can lead to a rapid homing of stem cells to the implanted sites during the first few days. To modulate cell differentiation and promote bone formation, slow and long‐term release of BMP‐2 from the scaffold is required. To achieve this goal, BMP‐2 can initially be loaded into particle delivery systems (e.g. microspheres), and these particles are subsequently introduced into the inner structure of the scaffold; such a design ensures the release of drug for as long as several weeks. In vivo data have shown promise that the sequential and controlled release of these two factors from well‐designed biomaterials may be able to regenerate calvarial critical size defects in rats without cell delivery in vivo milieu, this chemokine tends to be present in an inactive state due to protease cleavage, normally by CD26/dipeptidylpeptidase‐4 (DPP‐4) and matrix metalloproteinase‐2 (MMP‐2). To ensure the targeting of more reparative cells to damaged tissue, Kanki and colleagues created an engineered SDF‐1 in an MMP‐2/DPP‐4‐resistant form, termed SSDF‐1(S4V), and they found that this version of SDF‐1 could not be inactivated and was highly stable. In contrast to SDF‐1, which may be cleaved by DPP‐4, direct injection of protease‐resistant SSDF‐1(S4V) into an injured rat heart recruited more reparative cells to the damaged heart, leading to a dramatic improvement in ventricular function and angiogenesis For e.g. during antineoplastic therapy) via inhibiting apoptosis and inflammation. Moreover, G‐CSF may participate in neural tissue repair through stimulating neurogenesis, indicating an important non‐hematopoietic function of this biofactor in situ tissue regeneration, beneficial outcome was achieved via either local injection of G‐CSF or the incorporation of this factor into the implanted biomaterial. Local administration of G‐CSF for 14 days induced circulating EPC mobilization and recruitment to the implanted small‐diameter heparinized decellularized vascular graft, thus facilitating the generation of endothelium in the graft and the inhibition of neointimal hyperplasia via controlled G‐CSF delivery G‐CSF, also termed granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), was initially developed for the treatment of neutropenia after cytotoxic therapy and is approved for use in patients to prevent infection‐associated complications (i.e. chemokine (C‐C motif) ligand 7), this growth factor mediates signalling functions, including the proliferative response, cell survival and chemotactic activities. SCF synergizes with other therapeutic agents, such as G‐CSF, to augment cell mobilization and homing in vivovia an autocrine pathway, the interplay between this factor and the tyrosine kinase receptor c‐kit plays pivotal roles in preventing vascular smooth muscle cell apoptosis, facilitating homing by circulating SCF(+) cells and developing the neointima with bone marrow‐derived c‐kit(+) progenitors via decreasing the activities of NF‐κB, ERK1/2 and p38, further inhibiting CSC recruitment into peri‐infarcted heart tissue e.g. dental pulp) in vitro expansion SCF is a membrane‐bound soluble growth factor that is expressed on HSCs in either the primitive or the mature state. Like the c‐kit receptor CCL7 , Sordi and colleagues showed that the released factors present in islet supernatants can stimulate chemotaxis of BMMSCs; this stimulation was further demonstrated to be largely regulated by chemokine (C‐X3‐C motif) ligand 1 (CX3CL1) and CXCL12 (SDF‐1α) The above discussion includes only a few examples of the currently investigated chemokines that demonstrate promise for inducing host stem cell recruitment. Although each signalling molecule may act on multiple cell types, stromal‐like cells in the bone marrow are more likely to be activated by substance P via CXCR4 in the bone marrow milieu is key for retaining HSCs in a quiescent state and maintaining a number of distinct cell populations, the molecular events involved in cellular chemotaxis and migration remain largely unknown Collectively, human BMMSCs show detectable chemotaxis towards a wide variety of chemokines, including CCL2/3/5/7/17/19–22/CCL25/CCL28, CXCL8–13/16 and CX3CL1 , 116. Spvia VEGF receptor 1 via VEGF receptor 2 occurs in response to CXCR4 antagonism following VEGF pre‐treatment. Furthermore, in VEGF‐pre‐treated mice, administration of a CXCR4 antagonist produced detectable stromal progenitor cell mobilization, whereas G‐CSF administration did not Regulation of the selective mobilization of subsets of progenitors from the bone marrow, such as endothelial and stromal progenitors, depends on the cytokine microenvironment, which modulates cell retention and proliferation. By disrupting the CXCR4/SDF‐1α axis, for example, G‐CSF stimulates HPC release into the bloodstream via a single intravenous administration of LLP2A‐Ale via the co‐delivery of silicon ions and growth factors, which may exert a synergistic effect on endogenous stem/progenitor cells In the last decade, much of our current knowledge on stem cell homing and accommodation has been limited to how soluble biofactors, such as chemokines and cytokines, influence resident cells. Strategies for the administration of chemoattractants and chemical signals systemically and/or at the desired destination are critically important for enhancing the endogenous regeneration process because the presentation and release of signalling factors make the site of injury and/or inflammation more attractive to stem cells and hence can dictate stem cell homing e.g. see Fig. in vivo milieu underscores the importance of considering not only the correlation among cargo release kinetics, optimal time‐points and cell recruitment efficacy but also other environmental factors such as ECM stiffness, architecture and composition, which can jointly impact stem cell fates. Indeed, new findings continue to generate concern regarding how biomaterial cues may control cell geometry and how a diverse array of biophysical factors may be transmitted into the cell in vivo environment with a suitable hierarchical structure and topography at the nanoscale level and with proper mechanical properties Recently, extraordinary accomplishments have been achieved in the development of biomaterial vehicles that are capable of incorporating and releasing selected chemokines for therapeutic tissue regeneration based on cell recruitment and homing via chemical modification and/or genetic engineering in situ tissue‐engineering strategies without transplanting ex vivo‐expanded stem cells, this strategy offers an important tool for studying or regulating in vivo cell homing processes and would be useful for the future design of endogenous regenerative approaches via the secretion of growth factors or various other cytokines that enhance migration of endogenous stem cells to the lesion site and improve their function for integration, angiogenesis and neovascularization in situ tissue regeneration.As an alternative to delivering chemical signals, ions or other chemokines through a biomaterial platform, augmented cell homing can be achieved by making the surfaces of the targeted cells more responsive to specific homing inducers, typically via the introduction of specific gene sequences prior to transplantation; once these cells live in an in vivo milieu, they may express factors that induce recruitment, subsequently promoting the homing of endogenous stem cells Systemic infusion is a typical and convenient strategy that maximizes practical aspects of repeated doses and minimizes the invasiveness of cellular therapy; however, this delivery mode cannot guarantee engraftment efficiency that is, they are either not adaptable to different type of cells or unable to accommodate a vast array of ligand molecules. Immense effort is currently focused on making genetic modification simple and safe. Using lipid vesicles, Sarkar and colleagues reported a simple method to transiently modify cell surfaces, resulting in the ability to efficiently immobilize molecular ligands on cell surfaces for targeting to the inflamed site following systemic administration in vitro. Further experiments demonstrated that such chemical modification enabled the systemic infusion and targeted recruitment of MSCs to an inflamed region in vivo in a CD11a‐dependent (and other ICAM‐1‐binding domain‐dependent) manner via the bloodstream Although modification of cellular binding sites may be a viable approach to facilitating cell homing to a tissue of interest, current strategies, such as gene transfection, are practically complex and have potential safety concerns in vitro and in vivo, and MSCs move in response to a CXCL12 (SDF‐1α) gradient in vitro chemotactic response of human MSCs to thymus‐expressed chemokine (TECK), or CCL25, is more than 10‐fold greater than that to CXCL12 (SDF‐1α), indicating that TECK is a more potent in vitro chemoattractant for MSCs in vitro SDF‐1‐directed migration potential for human MSCs is quite low in vitro cell adhesion, migration and survival; and ameliorates the cells’ capacity to survive and engraft at the site of interest via its downstream genes SDF‐1α and VEGF in vitro and in vivoin vivo and to control this process may bring us closer to reaching the original goals of stem cell therapy.For stem cells to successfully home to sites of injury, a sequence of coordinated and regulatory interplays between a cell and its microenvironment provides signs that the signal is reaching the cell along its journey. Chemokines are the most important factors that regulate cell migration In situ tissue regenerative procedures offer great potential for broader clinical application and, more importantly, would only rely on off‐the‐shelf scaffolds without the need for ex vivo‐manipulated stem cells, thus reducing the time and cost in comparison with the classical tissue‐engineering approach The rising incidence of tissue insult caused by trauma, infection and/or destructive disease has led to an increasing demand for new, safe, effective and practical therapies for clinical use; this situation is exacerbated by an increasingly ageing population. In this context, the last two decades have witnessed remarkable laboratory‐based and preclinical success in the development of tissue‐engineering therapies via proliferation) as a transient pool, and the local functions of these cells within the materials, particularly considering secretion, concerted differentiation and remodelling in situ tissue engineering in vitro migration models have been used to study cell chemotaxis; however, a discrepancy between primary and ex vivo‐expanded MSCs in terms of mobilization or homing has been identified based on the observation that the cell phenotype undergoes dramatic changes during the in vitro expansion phase in vitro environments, and we still lack a reliable approach for in vivo monitoring of cell trafficking to an injury site. Based on in vivo microscopy, labelled cells can now be tracked in an animal after transplantation. Intravital microscopy can visualize labelled cells in a given tissue or organ after surgical dissection, thus revealing the in vivo chemokine‐directed interactions between MSCs and the endothelium and the subsequent cell migration within the ECM in situ, and the actual roles of endogenously recruited cell populations in tissue regeneration remain unclear in vitro and in vivo model investigations will be rewarded with increased potential across the arena of stem cell biology and therapy Functionally, issues remaining to be addressed include the chemotaxis of reparative stem cells following material transplantation, the amplification of these cells , to therapeutically stimulate receptor complexes and cells, or as vaccines for the delivery of multiple immunomodulatory molecules in situ tissue engineering will continue to provide new and more effective and treatments to address the problems currently encountered in the clinic As the implantation of outside devices into the in vivo is a new therapeutic strategy that holds great potential for achieving functional tissue regeneration without the need for the delivery of ex vivo‐expanded cells. To exploit homing mechanisms therapeutically, we must identify the complex interactions and pathways underlying the cascades of cellular events involved in wound healing and regeneration. Additionally, suitable animal models for tracking and evaluating endogenous reparative cells in vivo must be established to reveal the chemokine signalling and activity underlying directed cell recruitment. For this strategy to be successful, further investigations are required to ensure that the recruited cells propagate and divide into appropriate cell types for regeneration. In conclusion, the regulation of stem cell homing as a new paradigm for regeneration is still in its infancy. Further optimization of current material platforms that deliver potent signals for stem cell mobilization and homing and a better understanding of the priming or upstream signals involved in functional in situ tissue regeneration are necessary before an effective therapeutic outcome can be translated into the clinic for patient health care.The local use of signalling molecules at the area of injury to drive stem cell homing All authors participated in the drafting, writing and revision of the manuscript and gave final approval for publication of the work.Not applicable.The authors confirm that there are no conflict of interests.


The first World Health Organization (WHO) global health sector strategy on hepatitis B and C viruses (HBV and HCV) has called for the elimination of viral hepatitis as a major public health threat by 2030. This study assesses policies and programmes in support of elimination efforts as reported by patient groups in Europe.In 2016 and 2017, hepatitis patient groups in 25 European countries participated in a cross‐sectional survey about their countries’ policy responses to HBV and HCV. The English‐language survey addressed overall national response; public awareness/engagement; disease monitoring; prevention; testing/diagnosis; clinical assessment; and treatment. We performed a descriptive analysis of data and compared 2016 and 2017 findings.In 2017, 72% and 52% of the 25 European study countries were reported to not have national HBV and HCV strategies respectively. The number of respondents indicating that their governments collaborated with civil society on viral hepatitis control increased from 13 in 2016 to 18 in 2017. In both 2016 and 2017, patient groups reported that 9 countries (36%) have disease registers for HBV and 11 (44%) have disease registers for HCV. The number of countries reported to have needle and syringe exchange programmes available in all parts of the country dropped from 10 (40%) in 2016 to 8 in 2017 (32%). In both 2016 and 2017, patient groups in 5 countries (20%) reported that HCV treatment is available in non‐hospital settings. From 2016 to 2017, the reported number of countries with no restrictions on access to direct‐acting antivirals for HCV increased from 3 (12%) to 7 (28%), and 5 fewer countries were reported to refuse treatment to people who are currently injecting drugs.The patient‐led Hep‐CORE study offers a unique perspective on the readiness of study countries to undertake comprehensive viral hepatitis elimination efforts. Viral hepatitis monitoring should be expanded to address policy issues more comprehensively and to incorporate civil society perspectives, as is the case with global HIV monitoring. Policy components should also be explicitly added to the WHO framework for monitoring country‐level progress against viral hepatitis. The same resolution charged WHO with examining “the feasibility of and strategies needed for the elimination of hepatitis B and hepatitis C with a view to potentially setting global targets” Global Policy Report on the Prevention and Control of Viral Hepatitis in WHO Member States presents findings from a policy survey completed by representatives of 126 national governments To reach the targets and achieve WHO's overarching goal of eliminating viral hepatitis as a major public health threat by 2030, governments around the world must first consider whether they have the necessary policies and programmes in place. Scant information is available regarding the current policy landscape. The 2013 Policy monitoring of national responses to major health issues has been approached in a variety of ways With no ongoing government or multi‐stakeholder policy monitoring process for viral hepatitis in place at the global or regional level, ELPA sought to fill a gap by implementing the Hep‐CORE study in the countries where it had member organizations. These patient groups were responsible for reporting information to the study team. ELPA's goal was twofold: (1) to help liver patient groups identify key policy shortcomings in study countries; and (2) to engage liver patient groups in a regional policy monitoring initiative that will help hold countries accountable to their pledges to work towards viral hepatitis elimination. The following article reports findings from the 2016 and 2017 Hep‐CORE surveys.2A research team based at the University of Barcelona and the University of Copenhagen implemented the Hep‐CORE study in consultation with a multidisciplinary study group of viral hepatitis experts and carried out two rounds of data collection: one from July to October 2016 and another from August to November 2017.The original study instrument, a 39‐item English‐language online survey , a web‐based data collection tool that enables responses to be saved and edited over the course of multiple sessions The study cohort was recruited through a purposive sampling process. We emailed an invitation to participate in the survey to one liver patient group in each of the 24 European countries where ELPA had members at the time of 2016 study recruitment. In countries with more than one patient group, we selected the most representative group that was involved in viral hepatitis advocacy. We also emailed the invitation to a liver patient group in Denmark, since the patient group was just about to join ELPA, and did so in 2017. The same 25 patient groups that accepted the invitation and contributed data in 2016 were invited to respond to the 2017 survey. Many of the same individuals completed the survey in both years, although personnel changes in some patient groups meant that in some cases different individuals represented those groups in 2017.The 2016 Hep‐CORE Report, published by ELPA in early 2017 Following the close of each round of data collection, we reviewed data and queried study participants via email about incomplete, inconsistent or unclear information. We compiled and descriptively analysed final data using Microsoft Excel. We reported 2016 findings in 3In both 2016 and 2017, the 25 European liver patient groups that received study invitations all submitted surveys, for a 100% response rate. Box Box 113.1In 2017, patient groups in seven countries (28%) reported that their countries have written national strategies for HBV, and patient groups in 12 countries (48%) reported the same for HCV (Table 3.2While patient groups in most study countries reported opioid substitution therapy (OST) to be available in all parts of the country in both 2016 and 2017, far fewer reported needle and syringe programmes (NSPs) to be available in all parts of the country – 10 (40%) in 2016 and 8 (32%) in 2017  in 2017, and liver enzyme testing in 14 countries (56%)  reported HBV testing availability outside of hospitals in 2017, with the same number of countries reporting HCV testing/screening availability outside of hospitals in that year though with slight variation for HBV and HCV (data not shown). Types of testing sites included general practitioner clinics , OST clinics  and NSPs . In 2016, study participants were asked about non‐hospital‐based HBV and HCV testing and screening in a different way, with separate survey questions addressing the general population and high‐risk populations. Eleven countries (44%) in 2016 were reported to have HBV testing sites outside of hospitals for the general population, and 16 (64%), for high‐risk populations. Regarding HCV testing, the reported numbers of countries with non‐hospital‐based sites in 2016 were 13 for the general population (52%) and 16 for high‐risk populations (64%).3.4In 2017, patient groups in 19 countries (76%) reported that HBV treatment is provided in prisons, up from 18 countries (72%) in 2016 .Click here for additional data file.


CREB pathway, through different molecular mechanisms. Through careful implementation and optimisations, the algorithms detected a weak signal used to predict whether an animal was treated with nortriptyline (77%) or escitalopram (67%) on an independent testing set. The results from this study indicate that the molecular signature of AD treatment may include a much broader range of genomic markers than previously hypothesized, suggesting that response to medication may be as complex as the pathology. The search for biomarkers of antidepressant treatment response could therefore consider a higher number of genetic markers and their interactions. Through predominately different molecular targets and mechanisms of action, the two drugs modulate the same Creb1 pathway which plays a key role in neurotrophic responses and in inflammatory processes. © 2016 The Authors. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics Published by Wiley Periodicals, Inc.Response to antidepressant (AD) treatment may be a more polygenic trait than previously hypothesized, with many genetic variants interacting in yet unclear ways. In this study we used methods that can automatically learn to detect patterns of statistical regularity from a sparsely distributed signal across hippocampal transcriptome measurements in a large‐scale animal pharmacogenomic study to uncover genomic variations associated with AD. The study used four inbred mouse strains of both sexes, two drug treatments, and a control group . Multi‐class and binary classification using Machine Learning (ML) and regularization algorithms using iterative and univariate feature selection methods, including InfoGain, mRMR, ANOVA, and Chi Square, were used to uncover genomic markers associated with AD response. Relevant genes were selected based on Jaccard distance and carried forward for gene‐network analysis. Linear association methods uncovered only one gene associated with drug treatment response. The implementation of ML algorithms, together with feature reduction methods, revealed a set of 204 genes associated with SSRI and 241 genes associated with NRI response. Although only 10% of genes overlapped across the two drugs, network analysis shows that both drugs modulated the  Major depressive disorder (MDD) is a pervasive psychiatric disorder characterized by a number of clinical symptoms including: persistent low mood, anhedonia, insomnia, low energy, feelings of guilt, and ideation of death or suicide. MDD is also associated with a range of social impairments, including educational and occupational problems and with an increased risk of developing systemic disease, such as cardiovascular disease and Type 2 diabetes , being 1 for disjoint sets and 0 for equivalent sets.https://portal.genego.com/) was used to explore the molecular association between genes lists obtained from the two binary comparison  and known pathways maps and functional networks. MetaCore™ scores and prioritizes networks and pathways based on the relevance of the gene sets uploaded. The software evaluates the magnitude of the intersection between the uploaded genes and the set of genes corresponding to a network module using different statistical metrics including P values and G‐scores. P values are calculated based on hypergeometric distributions and used to establish whether saturation with the genes of interest is higher than random. When exploring signaling cascades, this allows one to evaluate if a network contains any fragments of well understood  signaling pathways. The G‐score is another metric used by MetaCore™ that effectively modifies the Z‐score based on the number of the linear canonical pathway fragments contained within the network. A network highly saturated with reference genes and containing several canonical pathways will achieve a higher G‐score. In this study we have explored the top ranking networks by P‐value and by G‐score and reported the highest associated functional pathway. These pathways were also compared to understand potential dissimilarities between the two drug comparisons.MetaCore™ , chance levels were considered at 33.3%. The results show that support vector machines with InfoGain using naive discretization was able to classify animals by treatment group above chance levels and outperformed both ℓAcross all methods used, the results get progressively weaker as we reduce the number of predictive features. This suggests that the molecular perturbations caused by antidepressant drugs are sparsely distributed across a very broad genomic region, pointing at the highly polygenic nature of AD response. It is likely that AD drugs affect in excess of 5,000 genes by a very small amount, which may explain the generally weak results reported using traditional linear association methods. The strongest classification considering error terms was achieved with the inclusion of 10,000 probe‐sets. Below 1,000 features none of the methods was able to classify animals into treatment group above chance levels in a 3‐way multi‐class design.Differences between treated and control animals were also explored as part of a simpler, two‐class solution for each of the two drugs separately. The first binary analysis used mRNA expression levels to classify animals into nortriptyline or control groups while the second analysis classified animals into escitalopram or control groups. A summary of results for the nortriptyline versus control group is presented in Table The highest classification accuracy was achieved with an SVM using radial basis function and 30,000 probe sets (77%). The results are consistent with the multi‐class analysis both in terms of number of features likely to be involved and in terms of the statistical methods used. The second binary comparison was conducted on the escitalopram or control groups. The results of this analysis show that it was possible to classify animals above chance level (>67%). However, classification was only possible using feature reduction (the best results obtained using ANOVA) suggesting a less polygenic architecture than what observed with an NRI Table . This maThe top ranking features based on Jaccard distance were extracted for each of the binary comparisons. A total of 241 probe sets were uncovered from the nortriptyline (NRI) versus control analysis and 204 from the escitalopram (SSRI) versus control comparison. Examining the two sets of probes, only 20 probe sets overlapped across both (<10%) analysis. This finding suggests that the two drugs have distinct molecular mechanisms of action. Indeed, the different gene targets could provide clues on the candidate gene sets that could be used in the search for predictors of differential treatment response in future human studies. In order to gain further biological insight the two gene‐lists were carried forward for pathway and network analysis using MetaCore™.P‐value and G‐Score are reported. The first pathway, with a P‐value of 4.58 × 10−28 and G‐Score of 49.06, containing 14 reference molecules was a network centered on the CREB1 gene complex  suggesting that antidepressants may also act through HDAC inhibition [Covington et al., The top ranking probe sets extracted from the nortriptyline and control classification analysis were uploaded to MetaCore™ for pathway and enrichment analysis. A total of 241 probe sets from the NRI versus control comparison were matched to 206 genes in MetaCore™ database . First, networks were created using uploaded reference molecules as seeds and interactions with molecules in MetaCore™ database as edges. The top scoring networks by P < 3 × 10−03 was also uncovered  is significantly associated with development of glucocorticoid receptor signaling . Through different mechanisms of action, networks uncovered for both NRI and SSRI are strongly involved in inflammatory processes. The second network, ranked by G‐Score (G‐Score = 1963.40), is an ephrin receptor signaling pathway, centered on the c‐Src gene hub  and receptor (EPHA5) genes of ephrin‐A5, in cluster of genes potentially involved in SSRI treatment response [Ising et al., The same analysis was repeated for probe sets uncovered from the escitalopram versus saline classification analysis. A total of 204 probe sets were identified and matched to 181 genes in MetaCore™ database. The first two networks uncovered were both strongly associated with P < 1 × 10−100) was associated with cellular organization (P < 5.85 × 10−08) and biogenesis (P < 6.6 × 10−08). The pathway consists of 138 uploaded (seed) genes of which 127 interact directly with the apoptotic signaling by c‐MYC. This may further highlight a role for both classes of antidepressants in neuronal cell growth in addition to showing immunological effects.We further performed a canonical pathway modeling to explore ontology for enrichment Fig. . The topP = 1.095 × 10−2) and cell regulation and proliferation (P = 7.070 × 10−4). A network analysis of common objects suggest an association with pathway (P = 4.93 × 10−11) centered on the C‐Jun gene hub involved the regulation of cell developmental processes and a second equally scored network by P‐value associated centered on Ubiquitin (P = 4.93 × 10−11). Given that both pathways target CREB1 and that IκB is a targeting mechanism to ubiquitination and subsequent proteasomal degradation, the pathway seems plausible and relevant to the pathways uncovered from both analysis [Taylor et al., MetaCore™, allows the comparison of different analyses for similarities and differences. Common processes across the two analysis are related to potential inhibition of TFG‐β signaling  than for escitalopram (67%). The results are consistent with existing literature showing a stronger association with NRI's rather than with SSRI's. Indeed, the only significant result to date across animal and human studies of AD from the GENDEP study was found with several common polymorphisms within the Creb‐1) molecule. Irrespectively of the distinct gene sets associated with NRI and SSRI treatment, the results point at specific overlaps in which these AD response pathways converge. It is conceivable that differences in these AD response pathways could, in part, be responsible for the differential response to AD treatments. Additionally, the overlap in the AD response pathways could provide insight into important downstream molecules mediating biological processes thought to alleviate symptoms of depression, such as reduced inflammation and neurogenesis as well as being implicated in etiology of the disorder.With only a 10% overlap between probe sets significantly associated with NRI treatment and probe sets significantly associated with SSRI treatment, it would appear the mechanisms of action of two drugs are fairly distinct. Contrary to this, gene set enrichment analysis shows that both the predictors of NRI treatment and SSRI treatment include a network centered on the Cyclic AMP‐responsive element‐binding protein‐1  [Tao et al., Creb1, is involved in neuronal adaptive responses, neurogenesis and neuronal survival and has been associated with the effects of AD treatment [Ghosh et al., Creb1 and AD response. The role of Creb1 in immune function is also thought to be an important mechanism underlying the well‐established association between inflammation, AD treatment and MDD. Creb1 promotes anti‐inflammatory processes via a number of mechanisms including the inhibition of NF‐κB activity and induction of IL‐10 expression [Parry and Mackman, P‐value. Furthermore, pathway analysis highlights genes predicting NRI treatment are primarily under the influence of glucocorticoids , one centered on c‐Jun gene hub, involved in the regulation of cell developmental processes, and the other centered on Ubiquitin, important for protein degradation following oxidation.Predictors of SSRI treatment and predictors of NRI treatment were combined and analysed using gene set enrichment with the aim of highlighting converging biological pathways that underlie both AD response and the aetiology of MDD. Two canonical pathways were returned with equal significance  and responders to SSRI treatment. The study searched for genetic markers for early (2 weeks), late responders and remitters (by week 5). The SNP was associated with all three phenotypes [Ising et al., A pathway highly enriched with canonical fragments that includes several ephrin genes was also associated with escitalopram treatment. Animal models have shown that stress may remodel neuronal dentrites and spines in the hippocampus but that these can be rescued by fluoxetine treatment. In mouse, ephrins are highly expressed in adult hippocampus neurons and play a critical role in neuronal regulation and synapse formation and plasticity [Li et al., The study used a bottom‐up data‐driven approach to empirically test different statistical learning methods to capture a shallow and sparse signal at the molecular level driven by AD‐induced perturbation. These methods offer several advantages compared to traditional methods but were both computationally expensive and required careful parameter optimization to avoid over fitting. In this study, we opted to err on the side of caution, sacrificing potentially improved accuracy for robustness of results although this may have increased chances of Type‐II errors.The study explored transcriptomic profiles in a disease relevant brain region in MDD animal models, which may inform on potential molecular mechanisms involved in the modulation of AD response in humans. However, mice are not miniature human beings and therefore findings require replication in human studies. Indeed there are several aspects of the trait, including environmental interactions, that simply cannot be modeled in mouse. However, this study found support for findings reported from the MARS human pharmacogenetic association study suggesting a role for the ephrin family gene in SSRI treatment response as well as a clear role for inflammatory processes.Additional methods for features reduction could be explored as part of future studies, which may include SVM‐RFE + mRMR to help overcome some of the limitation of the RFE method which yielded disappointing results. However this highlights the complexity and structure of the signal we are trying to detect. Robust Feature Selection (RFS) and Iterative Feature Perturbation (IFP) could also have been tested [Nie et al., The results may also point at further higher order complex interactions and post‐translational modification that are not captured by microarrays or by the methods used and that should be explored further. Although the assumptions that differential gene expression is associated with drug action is consistent with existing literature, expression changes in brain regions other than hippocampus and in blood have escaped the current investigation [Malki et al., ephrin gene in response to SSRI as previously reported in a human pharmacogenetic association study.The goal of pharmacogenomics research is to one day be able to predict which dose, of what drug will work for which patient at what time. However, uncovering a biosignature or genetic predictors of sufficient clinical utility to inform prescription of antidepressant drugs for an individual patient remains elusive [Tansey et al., Additional supporting information may be found in the online version of this article.Supporting Information.Click here for additional data file.Supporting Information.Click here for additional data file.Supporting Information.Click here for additional data file.


LTRs) of specific mouse endogenous retroviruses (ERVs) drive the expression of many long non‐coding transcripts (lncRNA). This process occurs post‐mitotically predominantly in spermatocytes and round spermatids. We demonstrate that this transposon‐driven lncRNA expression is a conserved feature of vertebrate spermatogenesis. We propose that transposon promoters are a mechanism by which the genome can explore novel transcriptional substrates, increasing evolutionary plasticity and allowing for the genesis of novel coding and non‐coding genes. Accordingly, we show that a small fraction of these novel ERV‐driven transcripts encode short open reading frames that produce detectable peptides. Finally, we find that distinct ERV elements from the same subfamilies act as differentially activated promoters in a tissue‐specific context. In summary, we demonstrate that LTRs can act as tissue‐specific promoters and contribute to post‐mitotic spermatogenic transcriptome diversity.Spermatogenesis is associated with major and unique changes to chromosomes and chromatin. Here, we sought to understand the impact of these changes on spermatogenic transcriptomes. We show that long terminal repeats ( The production of high‐quality gametes is essential to the propagation of life and the long‐term health of a species. Thus, cells of the germline and the molecular processes occurring within them carry special importance to the evolution of life. Spermatogenesis Fig A is a degag, pro, pol and env) flanked by two long terminal repeats (LTRs) that are essential for their replication env protein is no longer required and may be lost, while the replicative components of ERVs retain the hardware required to copy‐paste themselves to novel locations Transposable elements (TEs) occupy a large fraction of mammalian genomes having colonized approximately 35–50% of human and mouse genomes Non‐LTR retrotransposons, such as LINEs and SINEs, are also prevalent env genes play an essential function in placental development and ERVs are enriched within enhancers, contributing transcription factor binding sites Due to the features associated with their LTRs, ERVs are increasingly seen as both drivers of genome architecture but also as active players in shaping transcriptomes in tissue‐specific manners. ERVs have been shown to act as enhancers lncRNAs have previously been postulated as a possible pool for deriving novel functionality and novel peptides de novo transcript genesis.Previous studies of the male germline reveal a highly complex and global RNA regulatory network of mRNAs, lncRNAs and piRNAs with TEs and pseudogenes acting as regulatory sequences in vitro‐cultured spermatogonial stem cell lines, meiotic spermatocytes and the haploid round spermatids , and more than 75% of the lncRNA transcripts are novel  tags, we explored the 5′ localization of ENCODE 10(FPKM) values, although in raw expression terms, lncRNAs are still expressed at a lower level. This is in stark contrast to erythroblast controls, where lncRNAs expression is significantly lower than coding genes, in agreement with previously published data We inspected exon conservation across the assembly. Protein‐coding transcripts exhibit higher sequence conservation across their exons and in particular at splice junctions Fig C, while We sought to determine the genomic origins of this class of upregulated spermatogenic lncRNAs. Spermatogenesis coincides with dramatic chromatin remodelling associated with derepression of certain TEs, although transposition is suppressed at the post‐transcriptional level P < 1 × 10−42). This relative increase in the proportion of non‐coding promoters associated with LTR elements is also reflected as a genomewide enrichment for the elements in the promoters and at the TSSs of lncRNAs expressed in round spermatids, particularly at higher expression thresholds  and zebrafish (D. rerio). Rat diverged from mouse approximately 30 Mya Having observed ERV families driving expression of lncRNAs in spermatocytes and spermatids, we sought to determine whether this is a conserved feature of vertebrate spermatogenesis. We obtained and analysed data from rat  would evolve that could be translated into peptides. These emerging nascent ORFs would be subject to positive selection as they test, acquire or refine novel functionality. To test this hypothesis, we identified the longest ORF in each assembled protein‐coding and non‐coding transcript cluster. Subsequently, for each ORF, we calculated the fraction of bases undergoing negative selection or rapidly accumulating changes in the ORF compared to its putative 3′UTR. As expected, ORFs of protein‐coding transcripts show a greater proportion of bases undergoing negative selection Fig A as compet alOur results indicate ERVs are drivers of major transcriptional plasticity in the male germline. Previously, Faulkner Having established that ERV elements are expressed across many tissues, we wished to expand this analysis further to explore whether ERV‐regulated lncRNA expression is phenomenon‐restricted to the germline. Using Ensembl annotation and mouse ENCODE RNA‐Seq data, we identified tissue‐specific lncRNAs in a panel of 13 tissues. For these genes, we assessed the absolute number and fraction of transcripts with a promoter or TSS‐associated ERV element . From thThese results indicate that although the germline is a significant source of ERV‐driven transcription, ERVs can drive transcription across many tissues in a highly regulated manner.We present one of the most comprehensive analyses to date of non‐coding transcription during spermatogenesis, complementing the work of previous studies Naip genes is an ORR1E element, in contrast to the human copy of the gene As previously described we note an increase in promiscuous transcription in spermatocytes and round spermatids The activity of transposable elements is regulated by a number of mechanisms, and the onset of LTR‐driven lncRNA expression coincides with genomewide loss of the euchromatic H3K9me2 repression et alde novo gene genesis and subsequent selection of ORFs within lncRNA transcripts. By connecting ERV promoters presented here to extensive annotation of associated lncRNAs, we were able to investigate whether these transcripts could be further selected for function. As a first step, we have begun to explore their potential for the evolution of protein‐coding loci. Genesis of functional protein‐coding loci from intergenic regions and lncRNA has been demonstrated de novo genesis of both lncRNAs and peptide‐encoding loci.The germline is conceptually an ideal location to select and evolve transcript function, as any broadly toxic gene product (RNA or protein) would be rapidly eliminated through deleterious effects on gametogenesis. This “out of the testis” hypothesis has been described previously by Kaessmann et alIntriguingly, our analysis of FANTOM5 CAGE data demonstrated that LTR‐driven transcription can be far more widespread than we were expecting. This is in agreement with the work of Faulkner Finally, we can show that retrotransposon‐driven expression is a conserved feature of vertebrate spermatogenesis and plays a particularly significant role in driving and regulating lncRNA expression in rodents. Understanding the extent to which ERVs rewire transcriptional networks will be an important future direction, but this work helps to transfer ERVs from their traditional status as parasitic and opportunistic DNA elements to promoter elements with a major influence on the regulation, diversification and evolution of vertebrate transcriptomes.For full detail of experimental methods in all cases, please refer to the in vitroin vitro as described previously Ex vivo germ cells were obtained from dissection of 8‐week‐old adult mice through enzymatic digestion and mechanical disaggregation in EKRB buffer as previously described ® strain purchased from Charles River. Mice were maintained at the EMBL Mouse Biology Unit, Monterotondo, in accordance with Italian legislation  under licence from the Italian health ministry. RNA was prepared via Qiazol lysis followed by DNase treatment in the presence of an RNase inhibitor. RNA was recovered via EtOH precipitation; 5 μg of RNA was ribodepleted using Ribo‐Zero (Illumina), and 50 ng of this RNA was used for strand‐specific cDNA library preparation . Libraries were purified and analysed on High‐Sensitivity DNA chip (Agilent) on BioAnalyzer, and each paired‐end library was sequenced on one lane of a HiSeq 2000 sequencer.Per each cell type and species, libraries were prepared from two biological replicates. Erythroblasts were differentiated from E12.5 mouse livers Two biological replicates from erythroblasts, spermatogonial stem cells, spermatocytes and round spermatids were processed. Adapter contamination was removed, replicates merged and sequences de‐duplicated such that each sequence is unique per sample using Kraken −10) versus Ensembl Transcripts were assigned as non‐coding if they had a PhyloCSF Repeat annotation was obtained using RepeatMasker, NCBI/RMBLAST and the RepeatMasker database of elements (v20130422) for mouse, rat and zebrafish. When non‐overlapping repeat analysis is performed, the lowest scoring repeat match at overlapping sites is trimmed until no overlaps remain. Promoter analysis was performed by searching for overlaps between repeats and sites 1 kb upstream of defined TSSs. Unless otherwise stated see , when noP‐values were adjusted according to the Hochberg method. For comparison of coding to non‐coding promoter proportions, briefly, repeats were divided according to family, subfamily or class. Overlapping repeats in each set were merged. Expression was determined as above, and for each cell type, a minimum threshold of 0.25 FPKM was used. Promoters with non‐strand‐specific overlaps were merged. These promoter regions representing solely coding or non‐coding genes were retained. Promoter regions overlapping the repeat sets were counted. Repeat sets with fewer than 50 representative promoter regions were discarded. A hypergeometric test was performed to find repeat sets with a non‐coding/coding promoter region enrichment. The mouse ENCODE tissue analysis was performed by mapping 13 paired‐end strand‐specific samples to the mouse genome followed by count quantitation, normalization and generation of FPKM values where necessary (see Zebrafish and rat annotation was downloaded from Ensembl (v81), filtered and assigned a coding potential see . Gene exsary see .P ≤ 0.05) for both the ORF and paired 3′UTR.Detection of conserved lncRNAs was performed via mapping the longest isoform from each lncRNA cluster against 15 genome sequences obtained from Ensembl using WU‐BLAST swan (Kraken), and reads that map to a repeat masked genome were discarded . Remaining CAGE tags were mapped to a database of sequences for individual, non‐redundant ERV elements . Read depth was split between multimapped reads. Both uniquely mapped and multimapped counts were used.Cap analysis gene expression tags were retrieved from FANTOM5 A database for LC‐MS/MS search was created from a set of ranked ORFs selected based on a series of criteria see . SpermatPRJEB15333) and the European Bioinformatics Institute (http://www.ebi.ac.uk/research/enright/testome).Primary sequencing data, assembled transcripts and ancillary data are available via the European Nucleotide Archive (Study: MPD led the analysis of the data and performed the core computational analyses. CC contributed to the design analysis and generation of the library/preparation of the samples. HKS contributed to the analysis and the generation of the assembly. SvD, TL, GB, JMM and AJE contributed to the analysis. TA performed the mass spectrometry under the guidance of RCA and JR. AS and AB contributed to the analysis and provided the zebrafish samples. AJE and DO conceived and supervised this study. MPD, AJE and DO wrote the final version of the manuscript.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Review Process FileClick here for additional data file.


Mass spectrometry (MS) applications for intact protein complexes typically require electrospray (ES) ionization and have not been achieved via direct desorption from surfaces. Desorption ES ionization (DESI) MS has however transformed the study of tissue surfaces through release and characterisation of small molecules. Motivated by the desire to screen for ligand binding to intact protein complexes we report the development of a native DESI platform. By establishing conditions that preserve non‐covalent interactions we exploit the surface to capture a rapid turnover enzyme–substrate complex and to optimise detergents for membrane protein study. We demonstrate binding of lipids and drugs to membrane proteins deposited on surfaces and selectivity from a mix of related agonists for specific binding to a GPCR. Overall therefore we introduce this native DESI platform with the potential for high‐throughput ligand screening of some of the most challenging drug targets including GPCRs. A range of applications, including 2D imaging of small molecules and metabolites released from tissue cross‐sections, has become possible with the introduction of powerful DESI approaches when coupled with MS.N‐acetyl‐glucosamine (NAG) substrate added to the desorption spray and directed at lysozyme deposited on the stage we observed additional peaks assigned to binding of intact NAG‐5 to lysozyme  and the GroELm) containing octyl glucoside (OG) micelles and directed the desorption plume at the deposited protein. Initially a relatively low intensity signal was observed  0.05 % w/v) into the desorption plume induces a shift to higher charge states , a trimer of transmembrane beta‐barrels. We deposited 0.4 nmol of OmpF in ammonium acetate 200 m00 mm conVibrio splendidusScreening for optimal detergents, as part of membrane protein purification protocols, is time consuming and uses valuable protein resources.Pseudomonas aeruginosa, which translocates ferric‐pyoverdine (Pvd) across outer membranes,Turing to ligand screening an important criterion to establish is the extent to which protein complexes dissociate into their components during native DESI as opposed to conventional nanoflow ES. Selecting an outer membrane protein receptor FpvA from m) and deposited these protein‐peptide complexes onto the native DESI stage. The relative intensities of bound to unbound protein were extracted and plotted as a function of OBS1 concentration . The Kd determined (0.7±0.34 μm) for this membrane protein complex is in agreement with values reported (1.0±0.1 μm).Exploring further the quantitative aspects of this native DESI platform we selected OBS1 a 17‐residue peptide known to bind within the pores of OmpF with binding constants determined previously by both ITC and nanoES MS.apo OmpF (20 μm) was deposted and OBS1 added to the desorption buffer. Up to three peptides bound per trimer were detected with the predominant peak corresponding to two . This experiment confirms that the peptide can access binding sites within the short time frame during desorption, even in the presence of detergent. Similarly we added phosphatidylglycerol lipid (POPG) to the desorption buffer, at a concentration of 5 μm, and observed up to three lipids bound to OmpF . OmpF is also reported to bind to a range of antibiotics within the extracellular and periplasmic pore vestibule.m) to the desorption buffer reveals binding of one molecule of the antibiotic per OmpF trimer . Since we anticipate that the peptide binds within the pore, while the lipids bind to the outer surface and kanamycin to the top of the pore, we have highlighted our capability to bind directly to the membrane protein via three different mechanisms. In each of these three scenarios we assume that the small molecule has not reached solution phase equilibrium, since desorption is rapid, but rather has penetrated to some extent the protective micelle that surrounds the membrane protein while deposited on the surface.Beyond peptide binding established in solution we wanted to perform binding experiments on membrane proteins deposited on the stage. Delipidated 1, responsible for platelet aggregation and a key target for anti‐thrombotic therapy.apo form we added a cocktail of antagonists/agonists designed to target related GPCRs . These results reveal that this native DESI platform is capable of detecting selective binding of a specific antagonist to a GPCR from a multicomponent mixture.Building on this ability to screen for binding to a membrane protein target deposited on a surface we reasoned that it would be possible to add multiple ligands simultaneously. To explore this possibility we selected a Class A G‐protein couple receptor (GPCRs) depositing onto the stage 0.4 nmoles of P2YIn summary we have developed and applied a native DESI platform and shown that it is capable of preserving the native structure of both soluble and membrane proteins and their complexes. Comparing our approach with ambient ionization methods described previously we note the addition of chemicals in the spray solution in reactive DESI applications for small molecule analyses.The most exciting aspect of this native DESI approach however, is the potential of our method to study intact membrane proteins and their complexes. The ability to place membrane protein targets on planar surfaces in different lipidic environments, without tethering the proteins, and to carry out selective binding from a cocktail of drugs offers possibilities for high throughput screening. Many downstream applications become accessible including the ability to carry out multiple experiments on the same target; for example detergent optimisation, the screening of multiple lipids and ligands that bind to a drug target or the trapping of fast turnover products in enzyme catalysed reactions. Analogous to the powerful native MS methods, now widely accepted as a key component in structural biology, native DESI enables further possibilities for development of spatial, temporal and even directional analyses within artificial bilayers or membrane mimetics.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


This study investigated the impact of changing availability of tamper‐deterrent and non‐tamper‐deterrent oxycodone on prescribing patterns of controlled‐release oxycodone across Canada.We conducted a population‐based, serial cross‐sectional study of controlled‐release oxycodone dispensing from community pharmacies across Canada between October 2007 and April 2016. We calculated rates of dispensing (tablets per 100 population) and reported the relative market share of generic non‐tamper‐deterrent controlled‐release oxycodone. All analyses were reported nationally and stratified by province.After the introduction of a tamper‐deterrent formulation, the national rate of controlled‐release oxycodone dispensing fell by 44.6% (from 26.4 to 14.6 tablets per 100 population from February 2012 to April 2016). Between December 2012 and July 2013, there was moderate uptake of generic non‐tamper‐deterrent controlled‐release oxycodone , which appeared to have little impact on the overall rate of controlled‐release oxycodone dispensing in Canada. However, the uptake of generic non‐tamper‐deterrent oxycodone varied considerably by province. By April 2016, 55.0% of all controlled‐release oxycodone tablets dispensed in Quebec were for the generic formulation. Elsewhere in Canada, this prevalence was less than 30%, ranging between 1.6% (Prince Edward Island) and 26.9% (British Columbia) at the end of our study period.The changing availability of tamper‐deterrent and non‐tamper‐deterrent formulations of controlled‐release oxycodone in Canada has had variable influence on the rate of use of these products across Canada. Future research should explore whether the availability of generic controlled‐release oxycodone has led to measurable changes in the safety of oxycodone use in Canada. Despite fairly stable prescribing patterns in the 1980s and early 1990s, opioid prescribing rose dramatically in Canada following the approval of OxyContin,Since this time, policies have been introduced across Canada to address the rising use of oxycodone and its potential for misuse. These have included restricted access on some provincial drug programs and the introduction of safety warnings on product monographs.Despite these changes, and the associated controversies, little is known about the uptake of OxyNeo and generic controlled‐release oxycodone across the country. As policy‐makers and drug regulators debate the role that tamper‐deterrent opioid formulations can have in addressing opioid misuse and addiction, more information is needed to understand shifts in prescribing patterns when these drugs are reformulated. This study sought to determine the impact of changing availability of controlled‐release oxycodone formulations on rates of dispensing across Canada.2The introduction of a tamper‐deterrent formulation was associated with a 44.6% reduction in overall dispensing of controlled release oxycodone.There was moderate uptake of generic, non‐tamper‐deterrent oxycodone; however, this did not lead to an expansion in the overall prescribing rate of controlled‐release oxycodone.By 2016, 1 in 5 tablets for controlled‐release oxycodone that were dispensed in Canada were for a generic, non‐tamper‐deterrent form.Uptake of generic non‐tamper‐deterrent oxycodone was highest in Quebec, where more than half of all controlled‐release oxycodone is in this form.3We conducted a population‐based, serial cross‐sectional study of controlled‐release oxycodone dispensing from community pharmacies across Canada between October 2007 and April 2016. We used the QuintilesIMS Compuscript database to quantify prescription volumes for all long‐acting single‐agent oxycodone products dispensed over the study period, including those covered by provincial public insurance plans, private insurance companies and those purchased out of pocket. This database provides projections for prescription quantities at the provincial and national level based on data captured from a representative sample of approximately 6000 community pharmacies across the country. The geographic location of pharmacies, the distance between pharmacies and pharmacy size are all incorporated into projections, which are conducted by QuintilesIMS at the level of drug identification number. The quality of these data is continuously monitored and verified by QuintilesIMS, and the Compuscript database is regularly used for research purposes.4A total of 780.2 million tablets for controlled‐release oxycodone were dispensed across Canada over the study period, the majority of which  were dispensed in Ontario. In October 2007, the rate of controlled‐release oxycodone dispensing ranged from a low of 11.0 tablets per 100 population in Newfoundland and Labrador to 38.1 tablets per 100 population in Ontario  quickly replaced OxyContin, which was voluntarily withdrawn from the market by its manufacturerThe uptake of generic controlled‐release oxycodone varied considerably across Canada, with a range of 1.6% to 55.0% across the Canadian provinces in April 2016 Figure . In Queb5In this repeated cross‐sectional study of controlled‐release oxycodone dispensing patterns across Canada, we found reductions in the rate of dispensing of this drug after the introduction of a tamper‐deterrent formulation in February 2012. We observed no obvious “rebound” in national oxycodone dispensing after the introduction of generic non‐tamper‐deterrent products later in the same year. However, we did observe considerable variation in the market shares achieved by the generic forms across the Canadian provinces. The uptake was highest in Quebec, followed by British Columbia, Ontario, and Newfoundland and Labrador.The observed reduction in dispensing of controlled‐release oxycodone at the time of the introduction of the tamper‐deterrent formulation is aligned with changes in its listing on the majority of provincial drug insurance plans in Canada, which either chose not to list this new form of oxycodone on their formularies or listed it with exceptional access programs that required patients to meet a number of strict eligibility criteria before accessing the drug. Alberta, the only province to list OxyNeo as a full benefit on their formulary, had only minimal change in their dispensing patterns for controlled‐release oxycodone in February 2012 and was the province with the highest rate of prescribing by the end of our study period. This suggests that public drug funding and restricted reimbursement criteria influenced broad patterns of oxycodone use across Canada. However, these products have a relatively low cost, which might not deter their use in cases where a patient is prescribed the product by their physician but do not meet coverage criteria by a public or private drug insurance plan. Our study also suggests that the listing status of generic controlled‐release oxycodone had a significant impact on the uptake of these products. This is most apparent in Quebec where both brand (OxyNeo) and generic controlled‐release oxycodone are listed similarly on the drug formulary, with interchangeability and application of lowest prices in effect. This listing creates a financial incentive for the generic form of this drug to be dispensed, which may have led to the high degree of controlled‐release oxycodone dispensed in the province being for this non‐tamper‐deterrent form. Similarly, in British Columbia, the provincial drug formulary reimbursed the generic form until February 2015 when it was announced that the PharmaCare program would no longer cover these products.In Ontario and Newfoundland and Labrador, there was a relatively high uptake of generic controlled‐release oxycodone despite these products not being listed on the provincial drug formularies. In Ontario, where oxycodone has historically been the dominant long‐acting opioid on the market,5.1The core strength of this study is our ability to report changes in the dispensing patterns of all controlled‐release oxycodone prescriptions dispensed over more than 8 years across Canada at the national and provincial level. However, several limitations warrant emphasis. First, we did not have access to patient‐level data and, therefore, could not determine the degree to which generic controlled‐release oxycodone tablets were clustered at the individual level or rates of initiation of these products. Second, the Compuscript database only captures prescriptions dispensed from community pharmacies and, therefore, cannot comment on patterns of hospital use of controlled‐release oxycodone. Finally, we are not able to distinguish between publicly and privately funded  prescriptions. However, given that generic forms of controlled‐release oxycodone are generally not covered by public drug programs, this should have limited impact on our interpretation of these results.6Changes to the availability and listing status of generic and brand name formulations of controlled‐release oxycodone have led to important changes in both the rate of use of these products across Canada and the relative market share of non‐tamper‐deterrent opioids. Importantly, although the introduction of a tamper‐deterrent formulation in early 2012 led to considerable reductions in the rate of controlled‐release oxycodone dispensing, the marketing of generic non‐tamper‐deterrent formulations later that year did not lead to any subsequent expansion of the market. By 2016, 1 in 5 tablets for controlled‐release oxycodone that were dispensed in Canada were for a generic, non‐tamper‐deterrent form, and in several provinces, this was likely paid for by private insurers or cash payments. The geographic variation in uptake of these products suggests that provincial drug reimbursement policies—including restrictions on access through public drug formularies—has influenced these trends. In Canada, there was sufficient uptake of generic non‐tamper‐deterrent formulations of oxycodone to justify further research to determine if this has led to measurable changes in the safety of oxycodone use in Canada. This is important as drug regulators from around the world consider the role of tamper‐deterrent opioid formulations in addressing the opioid crisis and seek to understand how patent expiration and generic non‐tamper‐deterrent alternatives will impact the prescribed opioid environment.Tara Gomes has received unrestricted grant funding from the Ontario Ministry of Health and Long‐Term Care. No other authors report any competing interests.Appendix S1: Controlled Release Oxycodone Coverage Through Provincial Public Drug ProgramsClick here for additional data file.


OOP) costs incurred for medical and health needs by rural breast cancer survivors and what factors may be associated with higher OOP costs and the associated economic burden. Data were examined for 432 survivors participating in the Rural Breast Cancer Survivor Intervention trial. OOP costs were collected using the Work and Finances Inventory survey at baseline and four assessments every 3 months. Mean and median OOP costs and burden (percent of monthly income spent on OOP costs) were reported and factors associated with OOP costs and burden identified with generalized linear models fitted with over‐dispersed gamma distributions and logarithmic links (OOP costs) and with beta distributions with logit link (OOP burden). OOP costs per month since the end of treatment were on average $232.7 (median $95.6), declined at the next assessment point to $186.5 (median $89.1), and thereafter remained at that level. Mean OOP burden was 9% at baseline and between 7% and 8% at the next assessments. Factors suggestive of contributing to higher OOP costs and OOP burden were the following: younger age, lower income, time in survivorship from diagnosis, and use of supportive services. OOP costs burden rural breast cancer survivors, particularly those who are younger and low income. Research should investigate the impact of OOP costs and interventions to reduce economic burden.Little is known about out‐of‐pocket ( Patients with cancer, survivors, and their families incur considerable out‐of‐pocket costs for their medical and health needs regardless of healthcare insurance coverage The population of rural cancer survivors deserves some attention with regard to the OOP costs they may incur and the burden that these may cause. Rural cancer survivors report worse physical and mental health as well as being more likely to be unemployed due to health reasons than their urban counterparts The objective of this study was to report the OOP costs of rural breast cancer survivors who participated in the Rural Breast Cancer Survivor Intervention (RBCS) trial This study was approved by Institutional Review Boards of the University of Alabama at Birmingham and the Florida Department of Health. Participants were women living in rural Florida diagnosed with Stage 0–III breast cancer, within the first three years after completing primary cancer treatment, at least 21 years of age, and with telephone or cell phone access. Rural eligibility was established based on residence in one of 33 Florida rural counties, or in a rural pocket of one of 34 Florida urban counties A total of 432 RBCS participants were randomized to either the Early Education and Support intervention group or the Delayed Education and Support intervention group. The RBCS four components included one intake assessment; three education and support sessions; one follow‐up education and support session; and six support calls. The early and delayed interventions had the same components, but participants in the delayed group received the support calls in the first 6 months after enrollment, and the education and support sessions at month 7.OOP costs incurred by survivors for cancer‐related medical care and other care to maintain or improve well‐being were collected using the Work and Finances Inventory (WFI) questionnaire. The WFI is a descriptive 46‐item survey, adapted and modified from Given et al. To assess participants’ characteristics and treatment, we used the Breast Cancer Survivor Socio‐demographic and Treatment Survey which includes questions on socio‐demographics  and questions on breast cancer treatment  Data were collected at baseline and at 3‐month intervals for a total of five assessment points over 12 months. OOP costs were grouped as follows:Medical care, that is, expenses for hospital and doctor bills, emergency and usual care, medical supplies, prescriptions and over the counter drugs, physical therapy, and travel to the hospital, clinic, or doctor's office;Counseling and health maintenance, that is, expenses for family or individual support and counseling, nutritional counseling, genetic testing and counseling, alternative treatments such as massage, herbs, alternative healers, and gym or health club memberships or other expense related to exercise;Side effect management, that is, expenses for items such as wigs and prostheses;Home maintenance, that is, expenses for help received for house cleaning, cooking. additional home maintenance such as yard work, and additional child care; andOther OOP costs, that is, expenses related to insurance premium increases and other miscellaneous items.We used a repeated measure model fitted with generalized linear mixed methods and a gamma distribution to explore which factors were associated with OOP costs across all time points At each assessment point, we computed OOP burden as the percent of monthly income spent on OOP cancer‐related costs. To explore which factors were associated with OOP burden, we used a repeated measure model fitted with generalized linear mixed methods and a beta distribution with logit link using the same predictors listed above. Predicted means were calculated for those factors significantly associated with OOP burden.Six months after enrollment, 361 participants were retained (16% attrition). Due to an association between baseline self‐reported mental health and dropout risk Almost half of the 432 rural breast cancer survivors in this study were 65 years old and older, and the majority was married or living with a partner and Caucasian Table . About 2OOP costs per month measured at baseline, representing monthly costs since the end of treatment, were on average $232.7 (median $95.6) Fig. . The meaThe predicted mean OOP monthly costs were significantly higher at baseline $222.5) than at the other assessment points  was higher at baseline than in the other assessments (Out‐of‐pocket costs after completing primary cancer treatment remained a substantial portion of the income of rural breast cancer survivors. Most of these costs were for medical care, including for hospital bills and medications, and declined over time as women progressed from diagnosis and into survivorship. However, these expenses are large and represent almost 13% of income for women with incomes below $20,000. These findings lead to a strong rationale for developing and testing interventions to address financial burden.Cancer affects quality and quantity of life, and also the economic well‐being of survivors Our results are consistent with previous work showing that the financial toxicity of cancer does not end once primary treatment is over. Breast cancer survivors from Florida urban areas who had finished treatment and were within 2 years of diagnosis spent on average more than $300 per month in the period after diagnosis The rural breast cancer survivors most burdened by cancer costs were younger and low‐income survivors. These results are in line with others who found that these groups, as well as women of minority backgrounds, are more likely to report worse financial‐related outcomes. For example, they are more likely to report a financial decline since diagnosis Currently, to our knowledge, there are no effective interventions to address cost burden in cancer survivors. A recent systematic review summarized the rapidly evolving field of financial toxicity directly related to the continued and escalating costs of cancer care There are some limitations to consider. First, OOP costs were self‐reported and were not systematically verified. However, the data collectors encouraged participants to have receipts and bills on hand to jog their memory about expenses incurred. Second, we may have not included some costs that survivors considered related to cancer. For example, a group of survivors reported expenses for gifts to people who helped them as costs they would not have had if they were not diagnosed with cancer In this population of rural breast cancer survivors, the out‐of‐pocket costs of cancer‐related care persisted in the years post‐treatment and burdened especially younger and low‐income survivors. Research should continue to investigate the impact that costs of cancer may have on the ability of rural cancer survivors to afford follow‐up care and other services needed to optimize their well‐being, and develop interventions with a potential to address, at least in part, the long‐term economic burden of cancer.The authors report no conflicts of interest.