Cardiovascular disease (CVD) continues to be the leading cause of global morbidity and mortality. Heart failure remains a major contributor to this mortality. Despite major therapeutic advances over the past decades, a better understanding of molecular and cellular mechanisms of CVD as well as improved therapeutic strategies for the management or treatment of heart failure are increasingly needed. Loss of myocardium is a major driver of heart failure. An attractive approach that appears to provide promising results in reducing cardiac degeneration is stem cell therapy (SCT). In this review, we describe different types of stem cells, including embryonic and adult stem cells, and we provide a detailed discussion of the properties of induced pluripotent stem cells (iPSCs). We also present and critically discuss the key methods used for converting somatic cells to pluripotent cells and iPSCs to cardiomyocytes (CMs), along with their advantages and limitations. Integrating and non-integrating reprogramming methods as well as characterization of iPSCs and iPSC-derived CMs are discussed. Furthermore, we critically present various methods of differentiating iPSCs to CMs. The value of iPSC-CMs in regenerative medicine as well as myocardial disease modeling and cardiac regeneration are emphasized. Cardiovascular disease (CVD) remains the leading cause of death worldwide, killing 17 million people each year. The World Health Organization (WHO) estimates that by 2020 this number will reach 24 million. With complex multifactorial pathologies, including both genetic and environmental factors, CVD continues to be difficult to prevent. Current strategies against CVD include prevention  and pharmacological and/or surgical intervention. However, the effectiveness of drug treatment varies among individuals, while surgical interventions may not be applicable to all patients. New approaches need to be established to better understand the mechanisms of CVD and improve diagnostic and therapeutic strategies, particularly in the context of heart failure.Loss of myocardium results in the clinical syndrome of heart failure . The lonStem cell therapy (SCT) aims to reduce cardiac degeneration by regenerating cardiomyocytes (CMs) and is currently considered one of the most promising therapeutic strategies , 7. StemStem cells can be classified according to their “potency” or “differentiation potential” Table . ImportaAdult or somatic stem cells (ASCs) are non-embryonic multipotent stem cells found in the adult organism after embryonic development and residing in an area in tissues called the “stem cell niche” , 16. ASCESCs are isolated from embryos and can be classified as totipotent or pluripotent depending on their temporal existence during fetal development. Totipotent ESCs are present in the earliest eight-cell stage embryo, whereas pluripotent ESCs are found throughout the remainder of embryonic development. In this review, ESCs refer to the pluripotent type of ESCs, obtained from a 4- or 5-day-old embryo, also known as the blastocyst phase of development. ESCs are extracted from the inner cell mass of blastocysts and placed in a controlled culture that allows them to divide indefinitely without further cell differentiation. These ex vivo expanded cells serve as a paramount source of stem cells for transplantation therapies for many diseases, including cardiomyopathies, neurological disorders, and diabetes Fig. . HoweverEthically, the use of human ESCs (hESCs) is controversial, with many pro-life advocates being concerned about the isolation of hESCs from “living” embryos. In 2001, the USA government banned stem cell research by restricting federal funding for research on hESCs. To allow responsible scientific research involving human stem cells, the National Institutes of Health (NIH) established the “Human Embryonic Stem Cell Registry”, which lists 177 stem cell lines that are suitable for employment in federally funded research. Unfortunately, not all of these stem cell lines are readily available, and scientists have concerns about the quality and the longevity of these stem cell lines. To bypass these challenges, an increasing number of laboratories around the world are currently using iPSCs to limit the use of hESCs and the destruction of living human embryos.Practical considerations such as the availability of embryonic tissues and the isolation of relatively rare cell types have limited the large-scale production of pure stem cells for industrial and clinical applications. As such, the stem cell research field has explored other options, such as transforming fully differentiated adult somatic cells into pluripotent stem cell (PSCs). The reacquisition of a pluripotent state, known as “cell reprogramming”, represents a paradigm shift in our understanding of cellular differentiation and of the plasticity of the differentiated state.Xenopus tadpole and injected it into a recipient enucleated frog egg . Th. Th96]. Transgene-free iPSC lines were generated from human embryonic fibroblasts (hEFs), human embryonic kidney 293 (HEK293) cells, and adult skin fibroblasts using the PB transposon-based system . This apAlthough remarkable progress has been made towards safe and efficient reprogramming, the aforementioned methods involve integration of transgenes into the host genome with unpredictable interruptions to the host cell genome and downstream consequences. In order to avoid any permanent or transient genomic modifications a safer approach for iPSC derivation is to avoid both permanent and transient genomic modification. Therefore, non-integrating methods for cell reprogramming have been developed and considered.The viral non-integrating method involves the generation of iPSCs using non-integrating viruses such as adenoviruses and sendai viruses for the delivery of OSKM factors Fig. . As oppoNon-viral non-integrating methods consist of the derivation of iPSCs through virus-free and transgene-free techniques. This relies on the induction of iPSCs by transient transfection of plasmid DNA, minicircle DNA, or synthetic RNA encoding OSKM factors, as well as the direct delivery of recombinant proteins of OSKM factors into the cells.When transfected into cells, plasmid DNA replicates independently of the genomic DNA without incorporating into the genome of the host cells. Transgene-free iPSCs have been produced from mouse  and humaMinicircle DNA are small supercoiled derivatives of plasmids that are free of all prokaryotic vector sequences and are composed essentially of a small eukaryotic expression cassette (~ 4 kb). The absence of bacterial DNA backbone makes them powerful tools for genetic manipulation of mammalian cells. In addition, their small size enhances their transfection capacity and confers a long ectopic expression pattern compared to standard plasmids , 105. MiThe RNA-based method for somatic cell reprogramming consists of delivering OSKM factors by repeated administration of synthetic messenger RNA (mRNA), an approach that overcomes viral genome integration or immune responses to foreign DNA. Multiple human cell types have been reprogrammed using synthetic modified messenger RNA . FurtherThe protein delivery method involves the direct delivery of reprogramming factors  into the cell Fig. . ThroughNumerous chemicals and small molecules have been shown to improve the efficiency of iPSC generation or enable the reduction of the reprogramming factors required for pluripotency induction . These m(DNMT3B), and REX1 marked the fully reprogrammed state whilst alkaline phosphatase, SSEA-4, growth differentiation factor 3 (GDF3), human telomerase reverse transcriptase (hTERT), and Nanog are insufficient as markers. Recently, Burridge and colleagues claimed to have established culture conditions that circumvent the interline variability of iPSC lines, which could significantly facilitate the downstream characterization of the reprogrammed iPSCs and increase the number of suitable iPSCs for the needs of each project [Reprogramming of somatic cells is hindered by the heterogeneity of the derived iPSC lines, which affects their differentiation potential into specific cell lineages. Even a single reprogramming experiment could generate multiple iPSC lines which exhibit distinct molecular and functional characteristics –126. Thi project .The vast majority of studies on hiPSC derivation from somatic cells have employed dermal fibroblasts as the starting population for reprogramming –131. FibKeratinocytes, the most abundant cell type in the epidermis, are involved in the protection of the skin and provide strength to the hair and nails. One study has reported the generation of iPSCs from keratinocytes obtained from human foreskin biopsies and plucked hair . These cMelanocytes are skin-specialized cells responsible for the production of melanin, the darkening pigment of the skin. Similar to fibroblasts and keratinocytes, melanocytes have been derived from skin biopsies and expanded in vitro . When coThe major advantage of fetal neural stem cells is their ability to be reprogrammed using only the Oct3/4 factor . However+ cells isolated from freshly isolated or cryopreserved cord blood units have been reprogrammed to iPSCs using Oct3/4 and Sox2 [Cord blood cells (CBCs) have also been used to derive iPSCs. In fact, CD133and Sox2 . Anotherand Sox2 . CBCs caand Sox2 , mesenchand Sox2 , and endand Sox2 . This miand Sox2 , 70. Of + cells are a subset of stem cells with a therapeutic potential against multiple hematologic malignancies and immunodeficiency disorders. Cells expressing CD34+ are normally found in the bone marrow; however, the administration of some cytokines, such as the granulocyte colony-stimulating factor (G-CSF) and the granulocyte-macrophage colony-stimulating factor (GM-CSF) enhance their trafficking to the peripheral blood [+ to ~ 1% of the total cell count, offering an abundant source of progenitor cells for reprogramming [+ cells have been used as a starting population for iPSC derivation using the OSKM factors [+ cells is challenging. Furthermore, the intake of G-CSF for mobilization may lead to undesirable effects, especially in patients with cardiovascular diseases such as headache, nausea, and bone pain [CD34al blood . This prgramming . Periphe factors . So far,one pain .Adipose tissue is a specialized connective tissue derived from embryonic mesenchyme that contains a mixture of multipotent stem cells that have the potential to differentiate into multiple cell lineages, including bone, cartilage, and muscle , 143. AdIn spite of promising pharmacological and surgical interventions in CVD, heart transplantation remains the sole therapeutic option for end-stage heart failure. Alternative approaches may include the refurbishment of the CM population to rescue the failing myocardium and restore heart function. The derivation of CMs from hiPSCs is a novel therapeutic strategy that could transform the future of cardiovascular medicine. However, the establishment of differentiated CMs that fulfill this purpose requires a substantial improvement of hiPSC culture methods and CM differentiation. Various methods have been described to induce the differentiation of iPSCs into CMs. These methods are closely related to those traditionally employed for the derivation of CMs from hESCs, since hiPSCs and hESCs share similar characteristics and differentiation potential.In general, three small-scale PSC-to-CM differentiation strategies have been implemented: 1) embryoid body (EB) formation assays; 2) co-culture of undifferentiated PSCs with a visceral endodermal cell line (END-2); and 3) a confluent PSC monolayer in the presence of defined cardiogenic growth factors Fig. .Fig. 9InEB formation assays are the most common method to generate CMs from iPSCs. EB assays involve the growth of undifferentiated iPSCs as aggregates in suspension, causing them to form structures called EBs –147. ForVisceral endoderm is an extraembryonic cell layer formed in the early stage of embryonic development that secretes critical factors involved in embryonic development. Mummery and colleagues reported that co-culture of human and mouse PSCs with visceral END-2, derived from mouse P19 embryonal carcinoma cells, can efficiently induce their differentiation into CMs , 155. AlThis method consists of direct differentiation of iPSCs towards the cardiac lineage by sequential addition of defined growth factors known to induce cardiac development in various animal models. This sequential addition of specific growth factors aims to recapitulate, in vitro, the embryonic development of heart tissue. Nodal signaling in the ectoderm evokes mesoderm induction, thus marking the onset of gastrulation. The role of Nodal in the development of germ layers and the primitive streak is crucial. Indeed, loss of Nodal function has been shown to lead to loss of mesoderm and excessive ectoderm, as well as embryonic lethality during early gastrulation , 161. WhAlthough small-scale protocols are successful in producing a high percentage of iPSC-derived CM, they suffer from limited scalability, limited reproducibility, and heterogeneity. Moreover, large animal models, high-throughput assays, and tissue engineering need a constant supply of billions of CMs, which require, more advanced and scalable strategies. Large-scale production using 2D culture was successfully achieved by scaling out the culture surfaces. This approach, however, is not cost- or space-efficient. Therefore, mass production of PSC-derived CMs was successfully implemented using 3D industry-compatible platforms. Such platforms include matrix-dependent cultures, such as microcarrier suspension cultures and sphere culture with gellan gum polymer, and matrix-independent suspension cultures, including spinner flasks and bioreactors. Transition to 3D cultures require the generation of suspension aggregates from dissociated clumps, microcarriers, self-assembling aggregates, or forced aggregation by micropatterning. Maintenance of aggregates in homogenous conditions is achieved by rocking, agitating, or stirring the culture depending on the platform format being used. Multiple studies have successfully produced high yields of ventricular-like CMs in a large scale and from different hPSC lines using multiple chemical modulators and different bioreactors , 173. Ex2 tension) [Several approaches geared towards improving the differentiation and maturation of iPSC-derived CMs have been suggested. Some of these promising strategies include knockdown of certain genes , bioreactension) . This istension) .With WAVE bioreactors, however, wave-induced agitations and high mechanical loading led to sixfold lower increase in cumulative lactate dehydrogenase (LDH) with higher CM yields and faster kinetics compared to stirred tanks. Additionally, 97% CM purity by puromycin selection was achieved in 2 days  with WAVE bioreactors versus 7 days  with stirred tank cultures . These fAnother strategy consists of replacing the mouse embryo fibroblast (MEFs) feeder layer with human cells. Current methods of hiPSC culture involve the utilization of a feeder layer of MEFs. These inactivated MEFs are known to promote the proliferation of hiPSCs as well as to maintain them in an undifferentiated state. This, however, is not without the risk of exposing the cultured hiPSCs to animal contaminants. Attractively, however, and as has been published with hESCs, MEFs can be efficiently replaced by culturing autologous skin fibroblasts obtained from the same donor/patient , 186. AlIn an attempt to define the various molecules that could promote differentiation of iPSCs to CMs, a high-throughput screening system has been developed. Some of the identified molecules include resveratrol , vitaminIn summary, many factors play critical roles in influencing the differentiation of iPSCs to CMs. Some of these include the starting cell population, cardio-inductive molecules and growth factors, as well as culturing conditions. Empirically determined optimum employment of these factors is key for successful and efficient differentiation of iPSCs to CMs.Subsequent to differentiation, CMs need to be purified and enriched. To this end, several commonly methods are employed. These include the use of a pulled-glass micropipette for manual separation , densityIt is important to note that a major limiting step for SCT in cardiac regeneration is the purification and enrichment of stem cell-derived CMs. While several approaches for this goal have been employed, their efficiency remains somewhat debatable. There is an agreement, however, that for any such method of purification to be efficient, it ought to be fast, specific, and scalable with no genetic modifications. It is then that such a method can be viewed as a potential therapeutic approach for the use of iPSC-derived CMs in the cardiology clinic.Following purification, the iPSC-derived CMs need to be characterized to ensure they have the expected characteristics. The study of structure and function of iPSC-derived CMs is complicated by the fact that the differentiation method  and cult2+ fluorescent dyes and confocal laser scanning microscopy are commonly used to detect the presence of intact Ca2+ handling proteins and assess Ca2+ signaling in differentiated CMs [2+ transients) between host and differentiated CMs [Live cell imaging yields a large number of cellular measurements that can be used to monitor multiple aspects of cell structure and function. Ultrastructural analysis shows that hESC-CMs develop in vitro from spheroidal cells to elongated cells with a more organized sarcomeric pattern  Fig. 1010. Transated CMs , 222. Hiated CMs –225. Recated CMs . STED imated CMs . They arated CMs . Their fated CMs . Of noteated CMs . These cGDF3, REX1, and TERT in iPSCs during their differentiation into functional CMs [Among the different evaluation approaches, molecular biology, immunocytochemistry, qRT-PCR, and phosphoproteomic assays are also used to characterize the functional properties of iPSC-derived CMs. Immunocytochemistry uses antibodies that target specific peptides or protein antigens in the cell via specific epitopes. It is a valuable tool to detect the presence of gap junction proteins  at the borders of differentiated CMs where they mediate functional coupling with host CMs , 230. Imonal CMs , 233, anonal CMs , 234. Inonal CMs , 236. Phonal CMs , 238. Duonal CMs , 240.kr) channels results in the prolongation of the AP duration in ESCs and iPSC-derived CMs, whilst blockade of calcium channels results in the shortening of the AP duration [The electrophysiological properties of iPSC and ESC-derived CMs can be characterized using whole cell patch clamp, allowing measurement of action potential (AP) characteristics and specific cell membrane ion currents. Whole cell patch clamp is generally performed on individually isolated CMs. AP characteristics can also be measured from multi-cellular preparations using sharp impalement, or non-invasively using voltage-sensitive dyes. AP characteristics can be inferred from measurement of the field potential by plating individual cells or multi-cellular preparations onto multi-electrode arrays (MEA). MEA and voltage-sensitive dyes also allow AP conduction velocity and propagation patterns in multi-cellular preparations to be measured. AP recorded from iPSC and ESC-derived CMs with morphologies resembling nodal (pacemaker) tissue, atrial, and ventricular tissues have been widely described in the literature. It is unclear if this demonstrates distinct populations of cells committed to differentiation into these three subtypes of mature CMs or simply a population of immature CMs with a heterogeneous phenotype. Cells with “ventricular” AP, for example, often have a high degree of automaticity and an upwards sloping phase 4, which is more typical of nodal cells in adult myocardium. Interestingly, some groups report that the iPSC differentiation method seems to affect the electrophysiological phenotype Fig. . For insduration , 241. Chduration , 232.FigStructural changes following cellular differentiation can be assessed using analytical chemistry methods. Kawamura and colleagues analyzed N-glycan transition during iPSC-CM differentiation using HPLC-MS methods . Cell suIn summary, iPSC- and ESC-derived CMs appear to resemble the biochemical and molecular signatures of adult CMs, along with some of their structural and functional properties. However, some iPSC- and ESC-derived CMs retain the phenotype of immature myocytes. Whether this affects their utility in regenerative medicine or as disease models is not apparent and will be further discussed in the following sections.Disease modeling is an integral component of research efforts to understand the pathogenic mechanisms of CVDs and unveil promising therapeutic targets. Although large and small animal models have been extensively used for modeling human CVDs, they are expensive, ethically problematic, and their contribution to understanding human disease is arguably limited by their fundamental biological differences. hESCs, whilst also ethically problematic, have also been explored as CVD models by introducing causative human gene mutations into hESCs and inducing their differentiation into functional CMs in vitro. iPSCs have significant advantages over ESCs as they are derived from somatic cells, circumventing most ethical objections to ESC technology, and carry genetic mutations as well as any other modifier genes and contributing genetic factors, potentially facilitating recreation of patient-specific disease phenotypes in vitro.However, literature shows that iPSC-derived CMs are structurally, functionally, and genetically similar to early embryonic CMs . Despiteks) in ventricular patient-specific iPSC-CMs compared with controls (Table ks currents (Table kr) [kr was significantly reduced in patient-specific cells in which an increased sensitivity to arrhythmogenic agents was detected. The authors further tested the therapeutic effect of nifedipine (antihypertensive), pinacidil (vasodilator), and ranolazine  on the electrophysiological properties of diseased iPSC-CMs. The drugs were found to shorten AP duration and abolish abnormal depolarization (early after depolarization (EAD)). Similarly, Matsa and colleagues successfully derived CMs from related LQTS2 patients with KCNH2 mutation [iPSCs were first used to replicate a cardiovascular disease phenotype in vitro by Moretti and colleagues . They cols Table . In contts Table . Additioable kr) . The dermutation . Using pYazawa et al.  have alsAnother study examining the same disorder used iPSC-CMs to uncover the role of a different mutation in the sodium channels . SimilarOne of the technical problems encountered in this type of cellular assay is the phenotypic heterogeneity of the derived CMs between atrial, ventricular, and nodal cells, which express different AP patterns early after depolarizations. A recent study  proposedArguably, existing experimental tools are sufficient to model single ion channel disorders, and consequently the challenge is to leverage the potential of iPSCs to model more complicated disease phenotypes. One of the earliest attempts to do this was for the LEOPARD syndrome an autosomal dominant multisystem disorder resulting from a missense mutation in the PTPN11 gene that results in abnormalities of the skin, skeletal muscle, and cardiovascular system . The mosA further study, which created iPSC-CMs from DCM patients, demonstrated a different application from the previous studies for iPSCs in cardiovascular disease modeling . In thisDespite promising evidence that complex cellular phenotypes can be modeled using iPSC-CMs, questions remain. Several of these studies suggest that there was heterogeneity in the population of iPSC-CMs, or inclusion of non-CMs following the use of common differentiation techniques. This may limit the utility of iPSC-CMs for multicellular assays, which are the mainstay of molecular biological and cellular physiology, and consequently limit their utility as disease models. Some studies were able to recreate subcellular phenotypes in iPSC-derived CMs whilst failing to recreate DCM or HCM cellular phenotypes, suggesting that culture conditions or cell–cell interaction may be critical in developing disease phenotypes , 260. OvDrug development is expensive and high-risk , 262, wiCurrent therapies for heart failure are largely palliative, aiming to prevent the progression of heart failure and relieve symptoms . The onlCell therapy entails either mobilization of endogenous cardiac progenitor cells or transplantation of exogenous stem cells. Interestingly, these therapies are not mutually exclusive, and it has been widely suggested that cell transplantation promotes mobilization of endogenous stem cells . SeveralMost of the clinical experience had been with autologous CD34+ cell transplantation , 282. ThESCs and iPSCs, in contrast to many of the multipotent cell types used in clinical trials and animal models, are pluripotent and can differentiate into all cell types in the body, including CMs , 283–286Researchers have reported that transplantation of ESC-CMs into the infarcted hearts of rodents improve cardiac function , 290–294Novel potential applications of iPSC technology involve their use in combination with the zinc-finger nuclease (ZFN) technique to genetically modify patient-specific iPSCs . This teiPSCs have also been proposed as “biological pacemakers” in patients with acquired arrhythmia. The contraction of heart muscle is initiated by a syncytium of specialized cells (pacemaker cells) responsible for the generation of rhythmic impulses that control heart rate, which are propagated through the myocardium through specialized conducting fibers . This meiPSC-based cell therapy is not, however, without technical difficulties and these will need to be overcome before they can be readily applied in clinical practice. Firstly, reports suggest that undifferentiated iPSCs may elicit a significant host immune response . Whilst Srivastava and colleagues extended the concept of cellular reprogramming by demonstrating direct trans-differentiation of murine fibroblasts into functional CMs by retroviral delivery of three cardiac transcription factors: Gata4, Mef2c, and Tbx5 (GMT) . One weeA modified protocol has been used to directly reprogram mouse embryonic fibroblasts (MEFs) into beating CMs without an intermediate pluripotent state . Efe andThese studies suggest that differentiated somatic cells can be reprogrammed directly into functional CMs without transitioning first to an intermediate pluripotent state. Bypassing the pluripotent stage not only potentially makes generation of reprogrammed CMs much more efficient but significantly reduces the tumorigenic risk associated with the use of pluripotent stem cells. Nevertheless, these strategies still rely on viral vectors for reprogramming, which raises safety concerns and may limit their application in clinical practice.Over the course of only a few years since Yamanaka’s pioneering work on cellular reprogramming, the field of iPSCs has evolved dramatically. The lessons previously learned from ESCs enabled the rapid application of iPSCs to CVD modeling and cell therapy Fig. . The cha


With the recent publication by Silpe and Bassler , consideVibrio cholerae biofilm formation, resulting in biofilm dispersion  to take advantage of host motility to disperse to new locations.Induction despite sister lysogens, if limited in extent, could be viewed as a bet-hedging strategy (Avlund et al., en masse lysis of sister lysogens, reducing their potential to inactivate released virions (similarly, see synchronized lysis-inhibition collapse, If lysogens are locally exposed to equivalent levels of DPO, then this might trigger in situ timing of VqmAPhage-receptor expression and its ecological context, e.g., such as the spatial distribution of its or DPO's presence within phage VP882 lysogen microcolonies. Regardless of specifics, integrating host-derived signals presumably increases the environmental information obtained by a prophage. Perhaps, then, the timing and context of VqmAPhage expression can predispose DPO-motivated prophage induction toward circumstances which are more favorable to virion release than might be seen within dense populations of sister lysogens.Better understanding DPO-motivated prophage induction will require determining the All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Disruption of epigenetic regulation by environmental toxins is an emerging area of focus for understanding the latter’s impact on human health. Polybrominated diphenyl ethers (PBDEs), one such group of toxins, are an environmentally pervasive class of brominated flame retardants that have been extensively used as coatings on a wide range of consumer products. Their environmental stability, propensity for bioaccumulation, and known links to adverse health effects have evoked extensive research to characterize underlying biological mechanisms of toxicity. Of particular concern is the growing body of evidence correlating human exposure levels to behavioral deficits related to neurodevelopmental disorders. The developing nervous system is particularly sensitive to influence by environmental signals, including dysregulation by toxins. Several major modes of actions have been identified, but a clear understanding of how observed effects relate to negative impacts on human health has not been established. Here, we review the current body of evidence for PBDE-induced epigenetic disruptions, including DNA methylation, chromatin dynamics, and non-coding RNA expression while discussing the potential relationship between PBDEs and neurodevelopmental disorders. Across the world, humans face exposure to a vast number of industrial chemicals, whose potential for negatively impacting human health has long been a concern ,2,3. In Dysidea sp.) and found to have antimicrobial properties [PBDEs are a group of environmentally persistent chemicals that have been widely used as flame retardants on household consumer products since 1970s. Due to their environmental stability and propensity for bioaccumulation, PBDE concentrations have ubiquitously and cumulatively built up in our environment and in our bodies around the globe. Intriguingly, PBDEs enter the environment from both anthropogenic and natural sources. Historically, these compounds were first described in the biomedical literature as early as the 1960s—a decade before their anthropogenic production—when they were isolated from Australian marine sponges . All included studies were used for qualitative analysis. No quantitative meta-analysis was conducted. A major effort has been devoted to evaluating the potential risk of PBDE exposure to human health. For the sake of brevity, here, we will highlight some of the major points while pointing to relevant reviews and meta-analyses of the vast number of studies that have been published on the topic. Monitoring of environmental and human levels of PBDEs in the 1990s led to a rising general concern that they may be a serious human health risk, which was widely recognized by the early 2000s . At the Due to the persistent, widespread, and sometimes heavy exposure levels observed, much attention has been given to the roles of PBDEs in several major aspects of health: carcinogenicity, reproductive health, and disruption of hormonal signaling ,18,19. ISince the rise of concern regarding PBDE toxicity, several major impacted biological mechanisms have been identified and investigated. These and other less explored effects of PBDEs have been recently reviewed . BrieflyA recently expanding approach towards understanding PBDE toxicity has focused on PBDE-induced disruption of epigenetic regulation. Such mechanisms are interesting as potential PBDE targets as they would constitute a direct gene-environment platform for cellular disruption. Further, there is an expanding appreciation for the role of epigenetic mechanisms in neurodevelopment and cognition ,32, as wOne of the most commonly recognized epigenetic mechanisms is DNA methylation. Methylation commonly occurs at cytosine nucleotides positioned before a guanosine (CpG dinucleotides), resulting in a 5-methylcytosine. Given the extensive impact methylation has on transcriptional regulation , it comeStudies have assessed PBDE-exposure-dependent changes in global DNA methylation at various representative regions or at specific loci (promoters). Two of the most prominent examples of representative methylated regions include ALU elements and LINE1, which are common transposable repeats that can have adverse cellular impacts when de-repressed due to hypomethylation . RepetitOne of the earliest studies on the effects of PBDE exposure on global DNA methylation in humans found a negative relationship between measured BDE-47 levels and ALU %5mC in blood samples of healthy Korean adults, while not finding significant relationships for BDE-99 or LINE1 methylation . SimilarSeveral groups have also examined the relationship between PBDE levels and effects on the placental epigenome. In 2016, two reports were published on effects in human placental samples. In one, the authors made PBDE, PCB, DDE measurements in villous placental tissue samples and found positive associations of PBDE levels with IGF2/H19 imprinting and methylation status (bisulfite conversion and targeted pyrosequencing) and global DNA methylation (assessed by LUMA (luminometric methylation assay)) . In the Dmnt3ba expression in all generations [In addition to these human studies, PBDE-methylation relationships have also been investigated in model animals—mostly rodents—both in vitro and in vivo. In vitro studies have been conducted in different cell types, but consistently found negative correlations between PBDE exposure and methylation level. In the earliest of these studies, primary hippocampal neurons were exposed to various concentrations of BDE-209 for 24 hours and subsequently, a global decrease in DNA methylation was found by an antibody based ‘ELISA-like’ assay . Anothererations .To our knowledge, only two reports exist that found no relationship between PBDE exposure and DNA methylation levels of any targets measured in those studies. One found no detectable decrease in methylation at the p53 promoter after 24 hours of exposure to low micromolar doses  of BDE-47 in human neuroblastoma cells (SH-SY5Y), although activation of the p53 pathway in general was implicated in observed effects . The othOther reversible chemical modifications of chromatin include modifications to histone proteins that regulate chromatin structure and instruct remodeling processes, ultimately controlling gene expression ,59. StudHowever, there have also been a few studies that do report chromatin disruption following PBDE exposure. One study found that 24 h nanomolar range exposures to several PBDEs (BDE-47/99/153/183/209) induced micronuclei formation during cytokinesis in MCF-7 cells, an indicator of chromosomal damage occurrence preceding cell division . It has Oryzias melastigma) to BDE-47 led to sex-specific differential protein expression in male and female gonads, with several histone variants  being down-regulated in male gonads [Zea mays L.) to BDE-47, and its metabolites 6-OH-BDE-47 and 6-MeOH-BDE-47, led to elevated levels of ROS and phospho-H2AX, likely in response to DNA damage. Interestingly, the hydroxylated metabolite produced the most severe effects [There is also some evidence specifically for PBDE-induced dysregulation of histones and histone-regulating proteins. In an effort to understand the carcinogenic potential of BDE-209, the first such study found that HEK293T cells exposed to micromolar range levels of the toxin exhibited altered expression of chromatin-regulating genes, specifically a histone gene cluster that the authors hypothesize could affect nucleosome properties . In the e gonads . Another effects . Another effects . Aside f effects . Recentl effects .The potential importance of understanding the effects of PBDEs on chromatin dynamics cannot be understated given the fundamental importance of chromatin properties for regulating gene expression and thus cellular states. Going forward, it will be important for investigators to focus on identifying additional effects on chromatin while distinguishing those that are direct from indirect, hopefully allowing for elucidation of the underlying mechanism.Non-coding RNAs—such as long non-coding RNA (lncRNA) and microRNA (miRNA)—can also act as epigenetic regulators ,73. VariIn addition to these examples in humans, two rodent studies concerning non-coding RNAs in the liver have been published. The first found that BDE-47 exposure upregulates CYP3A1 in rat liver and that this upregulation is mediated by BDE-47-induced repression of miRNA-23b, which negatively regulates CYP3A1 mRNA via a 3’ UTR binding site . The othConsidering this growing body of work documenting epigenetic dysregulation induced by PBDE exposure, there appear to be several central lines of evidence emerging from research done in various health contexts, including: adipocyte differentiation and obesity, reproductive health—of both sperm/testes and the placenta, carcinogenicity , and negative impacts on nervous system formation and function. It is becoming clear that many, if not all, of these various aspects of human health are impacted by PBDE-induced disruption of normal epigenetic states and mechanisms.There are fairly consistent findings of a negative relationship between PBDE levels and DNA methylation from in vitro and non-human animal studies across varied cell/tissue types and methylation detection methods. However, the data from human samples is more difficult to interpret. Studies reporting effects on global DNA methylation levels inferred from representative regions have incongruent results, and evidence of alterations to methylation in the placenta are, likewise, not in direct agreement. However, this confoundment and the fact that human studies have so far been conducted across very different populations and models should only encourage further work on the topic, especially given indications from non-human animal and in vitro studies. It will be of great value if these types of studies can build on the tentatively established negative impact of PBDEs on methylation and begin to focus on understanding the mechanisms underlying the alterations, while continuing to clarify effects in human studies.Compared to DNA methylation, the literature is poorer regarding the effects of PBDEs on other epigenetic mechanisms such as chromatin dynamics and expression of non-coding RNAs. However, some interesting ideas are beginning to emerge. While not yet well understood, PBDE-induced dysregulation of histones and chromatin regulators is an intriguing intersection for PBDEs and neurodevelopmental disorders, bolstered by the recent emergence of chromatin regulation as a major node of NDD risk ,82. FurtGoing forward, a major challenge for epigenetic PBDE research will be to assimilate new findings into the existing framework of PBDE toxicity that has been established from insights into other major impacted biological mechanisms. It will also be important to carefully consider nuanced aspects of exposures including tissue and sub-cellular localization, conduct more research on environmentally relevant doses and mixtures of PBDEs, further explore the prevalence and effects of their metabolites, and, to the extent that it is possible, integrate evidence generated across human and non-human studies (both in vitro and in vivo). This will be necessary in order to construct a more wholistic understanding of how these compounds impact cellular states and, ultimately, phenotypic outcomes. Hopefully, with continued research, we may eventually be able to explain how and to what extent these pervasive environmental pollutants are related to the numerous human health conditions that they appear to be contributing to.


A common challenge with all opioid use disorder treatment paths is withdrawal management. When withdrawal symptoms are not effectively monitored and managed, they lead to relapse which often leads to deadly overdose. A prerequisite for effective opioid withdrawal management is early identification and assessment of withdrawal symptoms.The objective of this research was to describe the type and content of opioid withdrawal monitoring methods, including surveys, scales and technology, to identify gaps in research and practice that could inform the design and development of novel withdrawal management technologies.A scoping review of literature was conducted. PubMed, EMBASE and Google Scholar were searched using a combination of search terms.Withdrawal scales are the main method of assessing and quantifying opioid withdrawal intensity. The search yielded 18 different opioid withdrawal scales used within the last 80 years. While traditional opioid withdrawal scales for patient monitoring are commonly used, most scales rely heavily on patients’ self-report and frequent observations, and generally suffer from lack of consensus on the criteria used for evaluation, mode of administration, type of reporting , frequency of administration, and assessment window.It is timely to investigate how opioid withdrawal scales can be complemented or replaced with reliable monitoring technologies. Use of noninvasive wearable sensors to continuously monitor physiologic changes associated with opioid withdrawal represents a potential to extend monitoring outside clinical setting. Opioids are natural , semi-synthetic  or synthetic  narcotics primarily used to treat acute and chronic pain . They arOUD has significant societal impacts, such as escalating direct and indirect healthcare costs –9. FloreSeveral treatment paths exist for OUD including opioid maintenance treatment, detoxification (detox) , 12, andA common challenge with all OUD treatment paths is withdrawal management. Opioid users experience challenging and often severe withdrawal symptoms when they abruptly discontinue or reduce opioid intake. Intensity of opioid withdrawal symptoms depends on, among others, the type of opioid used, duration of usage, underlying medical conditions, and family history , 19. TimA prerequisite for effective opioid withdrawal management is early identification and assessment of withdrawal symptoms. One common assessment method for inpatient facilities  is patient observation. This method involves frequent patient observations  and depWhile opioid overdose deaths have increased tremendously over the past two decades such trend has been much more stable for chronic diseases, such as heart disease Fig. . In factand chronic heart diseases have leveraged and benefited from advancements in smartphone and wearable sensor technologies , population , intervention , and evaluation . Reviewers transferred abstracted data to a detailed Excel spreadsheet. Authors met and organized the information extracted from articles into type of withdrawal assessment method, symptoms monitored, and temporal window covered. For the survey-based methods, authors also extracted information on scale name, mode of scale/survey administration and rating criteria employed. Date of scale development was inferred from when the study was first published.Of the 21 articles that were included in the final review, 18 (86%) articles assessed opioid withdrawal using scales and surveys. Our search did not yield any empirical studies employing wearable sensors or mobile health  apps to monitor opioid withdrawal symptoms. The remaining 3 (14%) articles employed technology to detect physiological changes during opioid intake.The review revealed that opioid withdrawal scales are the main method of assessing and quantifying opioid withdrawal intensity. These scales employ a combination of observable behaviors (signs), patients’ self-reports (symptoms) and/or physiological measures. Overall, this search identified 18 different scales used over the past 80 years , Addiction Research Center Inventory (ARCI), Opiate Withdrawal Subjective Experience Scale (OPW), focused on subjective effects of opioid withdrawal. To further study these effects, Haertzen, Meketon, and Hooks  developeWith the emergence of methadone maintenance programs in the 1970s, physicians had a harder time establishing diagnosis of opiate dependence. Some patients mastered the art of going from one methadone program to the next, mimicking symptomatology of acute withdrawal to get a large initial dose of methadone. This widespread behavior caused a need for an objective scale to determine late-stage acute opiate withdrawal and withdrawal severity . ResearcA number of patient-reported outcome instruments have been developed to measure acute symptoms of opioid withdrawal symptoms. These instruments developed to measure withdrawal include OWS , SOWS 3, AdjectiSOWS-Gossop, Clinical Institute Narcotic Assessment (CINA) and Clinical Opiate Withdrawal Scale (COWS) are the most widely used instruments to evaluate opiate withdrawal symptoms in the reviewed articles , 22. SOWNine out of 18 available scales are clinician-administered only, 8 are patient-reported only, and 2 are both clinician-administered and patient-reported. Clinician-administered questionnaires are completed using traditional paper and pencil while clinicians talk face-to-face to patients and observe for opioid withdrawal signs. Patient self-administered questionnaires (patient-reported) require patients to complete questionnaires by hand and return them to the clinician. Scales that use both clinician-administered and patient-reported questionnaires  concurrently but independently require both patients and clinicians to use traditional paper and pencil to complete questionnaires. None of the reviewed literature reported on which mode of administration is preferred or provides utility.The scales vary in the number of scale items  included, ranging from as many as 550 scale items  to as few as 10 items . For example, the 550 items on the ARCI subjectively measure drug-induced effects as well as effects associated with personality and psychiatric disorders. The short-form of the ARCI has 49 items that allows for quick administration, and repeated measurements. On the other hand, OWS  is a 32-Scale items are made up of signs and/or symptoms. Whereas a symptom is a manifestation of a disease apparent to a patient alone, a sign is a manifestation of a disease that a clinician and/or patient can perceive . Thus, wThe scales differ in their use of rating criteria. For each item on a scale, various response categories are used to rate its clinical severity. Across all scales, ratings for items are summed up to create a total score, with a higher score indicating greater severity. Five  of the scales used binary rating systems . Three out of these 5 scales are clinician administered and the remaining 2 are patient-reported. Here, the total score for each scale is the sum of “true” or “present” items. Only 1 scale, SOWQ, employed a 20-item anchored analog scale. Patients rate each item on a 100 mm analogue scale anchored on both ends by pairs of pleasant and unpleasant feelings. The total score is the sum of analog scores for all 20 items .While most scales use Likert scale ratings, there is no agreement on the type of scale with four  out of 18 use a 5-point rating, 3  out of 18 use point-based rating, and 1 (ARSW) out of 18 scales uses a 10-point rating system. One scale, SDQ, uses a weighted 5-point rating system, for which physiological measures are given highest weights than behavioral signs, followed by patient reported symptoms. Many of the studies did not provide the rationale behind choice of points or weights for items on the scales.The scales also differ in the value of total score. For example, whereas scores for the 11-item CINA range from 0 to 31 and higher scores are associated with more severe withdrawal syndrome, scores for the 11-item COWS range from 0 to 47 with specific ranges for the level of severity .Temporal window covered differ among the scales depending, in part, on the nature of the legal substitute used  to precipitate withdrawal, mode of drug administration , and mode of scale administration. Five out of 18 scales, mostly patient-administered, have 24-h post-drug temporal window. Six scales require addicts to report their “immediate feeling”. The remaining scales varied markedly in temporal window covered, ranging from a 2-min observation period for COWS to 10, 20, 30 min post injection for the Wang scale.Three studies employed wearable biosensors to detect physiological changes during opioid intake. In all three studies, the biosensors were attached to the wrist as a band and the common biomarkers that were monitored included electrodermal activity (EDA), skin temperature, and whole-body acceleration. The temporal windows covered were before, during, and after opioid administration.The first of these studies, a preliminary observational one, employed a wristband biosensor  to continuously measure electrodermal activity (EDA), skin temperature, and physical movements of 4 patients before, during, and after cocaine and opioid administration. They found that patients with extensive opioid use demonstrated little to no change in EDA while low-moderate users of opioid showed rise in EDA and decrease in skin temperature . In a foThe scoping review of different opioid withdrawal monitoring methods revealed greater research efforts and emphasis on scales/surveys to monitor symptoms compared to technologies, which focused solely on symptoms associated with opioid uptake. While limited, advancements in non-invasive and wearable sensor technology could potentially serve as efficient complementary solutions in managing withdrawal symptoms either in inpatient or remote settings.Our results show that opioid withdrawal scales are and continue to be the main instrument used to assess and quantify opioid withdrawal symptoms. Over the years, scales have been developed either to assess the degree of physical dependence (before buprenorphine or methadone induction) and/or to compare the efficacy of opioid withdrawal treatments . HoweverCINA, COWS, and SOWS-Gossop appear to be the most widely used in research and clinical settings , 22. In Monitoring of opioid withdrawal with scales can be conceptualized as sampling of patients’ behavior or experience over time. However, not all signs and symptoms may be captured by questionnaires or retrospective reports of behavior . ReliancAnother issue is that opioid withdrawal scales that require subjective patients’ reports ask for recall or summary information over long periods of time. Patients’ contexts and mental states at the time of recall often bias their memory retrieval process . This exThe findings suggest that some opioid withdrawal scales may impose high length, complexity and frequency of administration to users. Lengthy questionnaires have been identified as a general obstacle in clinical practice  and may The current method of monitoring opioid withdrawal using scales is challenging outside of clinic or research environments. An opioid monitoring method that accurately monitors withdrawal symptoms as they occur in real time would provide several distinct advantages including the ability to obtain environmental and behavioral contexts surrounding withdrawal symptoms , additioUsing noninvasive wearable sensors, including temperature, accelerometer, electrodermal activity, and photo plethysmography sensors, to continuously monitor physiologic changes associated with opioid withdrawal represents a potential to extend monitoring outside clinical setting . SensorsEven though remote monitoring technologies have shown promise in the management of chronic diseases e.g., , 61), ap, ap61]),While sensor-enabled remote monitoring tools to assess signs and symptoms of opioid withdrawal shows potential, several important challenges remain to be investigated. Most importantly, patient engagement and compliance have consistently shown to be problematic in other domains utilizing such tools and technologies , 70, parThere are some limitations in the study that warrant discussion. The scoping review utilized relatively fewer, albeit relevant, number of databases to identify potentially eligible studies. Despite this limitation, we found saturation in the types and descriptions of subjective methods for opioid withdrawal monitoring . The limited findings pertaining to technology-based methods may likely be due to the lagging nature of technology development for opioid withdrawal and/or uptake monitoring. It is also possible that technology design processes are not effectively captured and disseminated using traditional peer-reviewed analysis approach. Indeed, to capture emerging trends in technologies for opioid withdrawal monitoring, non-empirical investigations like technology landscape analysis of commercial technologies  may be more appropriate .While traditional opioid withdrawal scales for patient monitoring are commonly used, most scales rely heavily on patients’ self-report and frequent observations, and generally suffer from lack of consensus on the criteria used for evaluation, mode of administration, type of reporting , frequency of administration, and assessment window. Therefore, it is timely to investigate how such scales can be complemented or replaced with reliable monitoring technologies. Smart sensing technologies that provide real-time information on the physiological and psychological status of patients have shown promise in addressing a similar need for other conditions such as diabetes, cardiovascular diseases, and mental health and may significantly, and proactively, improve treatment care of opioid patients by keeping clinicians aware of patient status.Work is in progress to identify viable technologies to assess a wide range of withdrawal symptoms more objectively. This includes a comprehensive systematic review and analysis of past, present and future trends in wearable sensor technologies. Our long-term goal is to develop a technological framework using a human-centered design process that is non-invasive, reliable, and proactive to detect and manage opiate withdrawal symptoms by utilizing an array of sensors that can be employed to detect temporal and spectral patterns of physiological and psychological responses associated with short- and long-term withdrawal symptoms.


The elusive nature of sudden unexpected death in epilepsy (SUDEP) has led to investigations of mechanisms and identification of biomarkers of this fatal scenario that constitutes the leading cause of premature death in epilepsy. In this short review, we compile evidence from structural and functional neuroimaging that demonstrates alterations to brain structures and networks involved in central autonomic and respiratory control in SUDEP and those at elevated risk. These findings suggest that compromised central control of vital regulatory processes may contribute to SUDEP. Both structural changes and dysfunctional interactions indicate potential mechanisms underlying the fatal event; contributions to individual risk prediction will require further study. The nature and sites of functional disruptions suggest potential non-invasive interventions to overcome failing processes. Sudden unexpected death in epilepsy (SUDEP) is the leading cause of untimely death in epilepsy , with a n = 10 cases). Specifically, transient cessations in breathing preceded terminal apnoea, and ultimately terminal asystole. Prolonged peri-ictal apnea  show autonomic and respiratory dysfunction preceding SUDEP. A comprehensive assessment of the incidence and mechanisms of cardiorespiratory arrests in EMUs  revealedal apnea , with oral apnea  may playal apnea , particual apnea . Sustainal apnea , 10 and al apnea .GTCS are the leading SUDEP risk-factor ; experieNeuroimaging is a powerful tool to explore structural and functional brain alterations within distinct sites and networks crucial for autonomic and respiratory regulatory processes in patients who (after being scanned) succumb to SUDEP. Such assessments allow the investigation of structural (tissue) abnormalities or disrupted networks related to SUDEP, and have the potential to shed light on underlying mechanisms and provide biomarkers to prospectively identify living patients at heightened risk. In the following sections, evidence from structural and functional magnetic resonance imaging (MRI) investigations into SUDEP will be discussed, together with potential interventions to overcome deficient processes.Structural MRI enables the identification and characterization of brain tissue abnormalities, regional alterations in brain volume, cortical thickness and morphometry, and abnormal structural connections (fiber tracts). Such techniques have been widely applied to epilepsy –17, and n = 12) and comparable high-risk, low-risk, and healthy controls (n = 25 SUDEP cases) which employed family-wise-error rate (FWER) correction of p-values. Thalamic volume loss in patients who experience GTCS, and therefore who are at greatest risk of SUDEP, has been widely demonstrated  has been used to investigate regional gray matter changes in subjects who later died from SUDEP  revealed widespread thinning, most prominently within the frontal lobe, including orbitofrontal sites, which are involved in cardiovascular regulation ].Overall, there is accumulating evidence of widespread structural loss, particularly within anatomic regions related to cardiorespiratory functions such as thalamic, frontal lobe  as well as brainstem sites in people who suffered SUDEP and in those at greatest risk.In addition to regional reductions, regional increased volume, and cortical thickness in key autonomic, breathing, and sensory sites have been observed in SUDEP cases and those at high risk. Compared with low-risk and healthy subjects, cases of SUDEP and those at high risk show increased gray matter volume of the right amygdala and anterior hippocampus , which an = 53) compared with healthy controls (n = 530). While patients who experience GTCS are at highest risk of SUDEP, assessments of cortical thickness are needed in patients who died from SUDEP, since studies including only at-risk populations are complicated by limited interpretability. Elevated volume and cortical thickness are traditionally considered as being linked to improved function or compensatory mechanisms, such as the increased volume within visual cortex observed in deaf vs. hearing individuals  demonstrates reduced volume and cortical thinning in thalamic (primarily within posterior portions), frontal , and midbrain/cerebellar/brainstem sites . IncreasResting-state (RS) fMRI is a brain imaging technique in which subjects undergo fMRI scanning while lying “at rest” in the sense that they are not subjected to any experimental stimulus or task; they are usually asked to lie quietly and stay awake, with their eyes closed. Although it has been argued that the “rest state” in question lacks specificity, this technique has the advantage of being applicable to a wide range of subjects, such as those incapable of performing a specific task  and thun = 25 patients. High-risk patients exhibited reduced FC between the pons and right thalamus, midbrain and right thalamus, bilateral anterior cingulate and right thalamus, and between the left and right thalamus. In another study Allen et al. , as well as between bilateral medial/orbital frontal cortices and bilateral insula cortex.To date, two studies using RS-fMRI have focused on the FC between brain regions related to central mediation of autonomic and respiratory processes in patients with epilepsy , which bring together investigators from institutions across the US and UK and utilize open data sharing, will be crucial. Also, the integration of multi-modal imaging data, acquired prospectively seems essential for improved characterization of the relationship between structural and functional brain alterations: diffusion MRI, RS-fMRI, and T1- and T2-weighted MR images to investigate how volume changes and structural and functional connectivity alterations among regulatory structures arise and change in relation to clinical manifestations such as seizure frequency and disease duration. Additionally, the availability of ever larger retrospective datasets (including genetic data) for the wider research community would benefit efforts to better characterize SUDEP  and establish biomarkers using data-driven approaches.Volumetric and morphological structural changes within the brainstem are a crucial aspect of research into SUDEP mechanisms, since the region contains many of the final common autonomic and respiratory pathways. Some anatomical properties of the brainstem and MR resolution limitations have restricted imaging studies to volumetric evaluation, either by gray and white matter segmentation, or amount of warping required to match a common template. Both techniques, which rely on T1-weighted contrast, may be insufficiently sensitive to detect underlying tissue changes within critical structures, particularly since many are small nuclei which lie on the border of white and gray matter. However, other newer procedures, such as quantitative MR T1/T2 ratio scans will enable assessment of myelin integrity, providing insights into allowing necessary evaluation of supportive tissue for neuronal processes in the brainstem and elsewhere.2 or hypoxia provocations, Valsalva maneuvers, cold pressor, or hand grip challenges may be useful to show magnitude of responses, timing delays or advancements between linked respiratory and cardiovascular areas. In other pathologic conditions, such as heart failure or congenital central hypoventilation syndrome, both distortions in timing and amplitude of linked structures appear (The observed disruptions of resting-state patterns in SUDEP patients mandate the assessment of failed vital functions, namely studies which incorporate concurrent recordings of autonomic and breathing patterns during fMRI scanning, enabling characterization of associations between resting FC and resting cardiovascular and breathing processes. In addition, conventional correlations of “evoked” fMRI changes to breathing and cardiovascular changes to triggered challenges, e.g., COs appear , 60. SucThe observed alterations in FC between brain structures which have the potential to elicit a cardiovascular or breathing crisis leading to SUDEP raise the issue of how 1 day we might be able to intervene in those dysfunctional pathways to avoid or overcome such crises. Potential targets for intervention are the neurotransmitters in the affected pathways or the enhancement of pathways for protective recovery circuitry. In addition, advances in neuromodulation procedures offer a means to intervene directly in disrupted functional pathways, which is of particular use here, since 31% of epilepsy patients are drug resistant . NeuromoPeople who succumb to SUDEP, and those at risk, undergo regional brain structural changes and resting-state fMRI alterations between essential areas regulating cardiovascular and breathing control, indicating a structural and functional basis for impaired communication between areas necessary for recovery from compromised vital circumstances. The findings, although limited in sample sizes, are sufficiently apparent that indications of structural and functional changes may signal risk for SUDEP and shed light on underlying mechanisms. Moreover, both the structural and functional outcomes suggest means for potential interventions with specialized pharmacologic or neuromodulatory procedures. The proper characterization of the respective roles of the known risk factors, such as GTCS and disease duration, in relation to imaging findings can contribute to understanding SUDEP mechanisms, and warrant further investigation to disentangle clinical factors from what may be related to SUDEP.LA and RH designed and prepared the manuscript. SL, LL, and BD contributed to preparation, critical review, and editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with one of the authors SL.


Scientific Reports 10.1038/s41598-018-26737-2, published online 05 June 2018Correction to: The original version of this Article contained errors in the Abstract.“We report on a novel kind of accelerating beams that follow parabolic paths in free space. In fact, this accelerating peculiar polygon beam (APPB) is induced by the spectral phase symmetrization of the regular polygon beam (RPB) with five intensity beam (RPB) with five intensity peaks, and it preserves a peculiar symmetric structure during propagation. Specially, such beam not only exhibits autofocusing property, but also possesses two types of accelerating intensity maxima, i.e., the cusp and spot-point structure, which does not exist in the previously reported accelerating beams. We also provide a detailed insight into the theoretical origin and characteristics of this spatially accelerating beam through catastrophe theory. Moreover, an experimental scheme based on a digital micromirror device (DMD) with the binary spectral hologram is proposed to generate the target beam by precise modulation, and a longitudinal needle-like focus is observed around the focal region. The experimental results confirm the peculiar features presented in the theoretical findings. Further, the APPB is verified to exhibit self-healing property during propagation with either obstructed cusp or spot intensity maxima point reconstructing after a certain distance. Hence, we believe that the APPB will facilitate the applications in the areas of particle manipulation, material processing and optofludics.”now reads:“We report on a novel kind of accelerating beams that follow parabolic paths in free space. In fact, this accelerating peculiar polygon beam (APPB) is induced by the spectral phase symmetrization of the regular polygon beam (RPB) with five intensity peaks, and it preserves a peculiar symmetric structure during propagation. Specially, such beam not only exhibits autofocusing property, but also possesses two types of accelerating intensity maxima, i.e., the cusp and spot-like structure, which does not exist in the previously reported accelerating beams with a single kind of lobes. We also provide a detailed insight into the theoretical origin and characteristics of this spatially accelerating beam through catastrophe theory. Moreover, an experimental scheme based on a digital micromirror device (DMD) with the binary spectral hologram is proposed to generate the target beam by precise modulation, and a longitudinal needle-like focus is observed around the focal region. The experimental results confirm the peculiar features presented in the theoretical findings. Further, the APPB is verified to exhibit self-healing property during propagation with either obstructed cusp or spot reconstructing after a certain distance. Hence, we believe that the APPB will facilitate the applications in the areas of particle manipulation, material processing and optofludics.”These errors have now been corrected in the PDF and HTML versions of the Article.


Viral encephalitis is a rare but clinically serious consequence of viral invasion of the brain and insight into its pathogenesis is urgently needed. Important research questions concern the involvement of the host innate immune response in pathogenesis, key to which is the role played by microglia, resident macrophages of the brain parenchyma. Do microglia have a protective function, by coordinating the innate immune response to viral infection, or do they drive pathogenic neuroinflammation? Here we synthesize recent data from mouse models of acute viral encephalitis, which reveal an unambiguously protective role for microglia. Depletion of microglia, via blockade of colony-stimulating factor 1 receptor (CSF1R) signaling, led to increased viral replication accompanied by more severe neurological disease and heightened mortality. Whilst the underlying mechanism(s) remain to be defined, microglial interactions with T cells and phagocytosis of infected neurones appear to play a role. Paradoxically, the production of inflammatory cytokines was increased in several instances following viral infection in microglia-depleted brains, suggesting that: (i) cells other than microglia mediate inflammatory responses and/or (ii) microglia may exert a regulatory function. Under certain circumstances the microglial antiviral response might contribute negatively to longer-term neurological sequelae, although fewer studies have focused on this aspect in encephalitis models. Understanding regulation of the microglial response, and how it contributes to disease is therefore a priority for future studies. Collectively, these findings demonstrate the central role of microglia in pathogenesis, suggesting the exciting possibility that defects of microglial function might contribute to encephalitis susceptibility and/or outcome in humans. Viral encephalitis is defined as pathological inflammation of the brain parenchyma secondary to viral infection . This syWhilst some viruses are highly neurovirulent , most people infected with neurotropic viruses do not go on to develop encephalitis, suggesting that rare host and/or viral factors underlie susceptibility. Defining these factors is a major challenge for the field. Despite decades of investigation, there is limited evidence of viral determinants of neurovirulence , 5, implThe hypothesis that encephalitis arises through a failure of host resistance is supported by very rare genetic errors of innate antiviral immunity, which confer heightened susceptibility to encephalitis secondary to wild-type –14 or athttps://clinicaltrials.gov/ct2/show/NCT03084783)  a lack of correlation between viral loads in cerebrospinal fluid and disease outcome –20, sugg3084783) .Mouse models of viral encephalitis provide an alternative means to resolve these issues. Whilst there are caveats to the use of mice to understand human disease , 29, keyContrary to the long-held view of the brain as an immune-privileged organ, it is now clear that the brain parenchyma poses an intrinsic antiviral network, in which the antiviral cytokines known as type I interferons (IFNs) play a central role , 33. DetIt has long been suspected that microglia—the sole brain-resident immune cells—play an essential role in antiviral defense of the brain . Howeverin vitro  have been used in a limited number of studies of viral encephalitis  underlying these protective properties remain uncertain. Preliminary data suggest that the T cell response induced by microglia is involved, however the impact on the quality and magnitude of the antiviral T cell response was inconsistent across different models –78, and Viral clearance and/or disease outcome relies on a functional T cell response in several mouse models of encephalitis . In the TMEV model, strain-specific differences in disease phenotype are linked to the effectiveness of the CD8+ T cell response, which is subject to Treg suppression, and consequent clinical disease, in susceptible mouse genetic backgrounds . AlthougIn MHV, both CD8+ and CD4+ T cells are involved in viral clearance , 81, whiThe collective implication of these studies is that microglia contribute positively to aspects of T cell recruitment and/or activation, however the precise nature of the protective T cell response induced by microglia requires further examination.Il34LacZ/LacZ mice were included in this study to show that the attrition of presynaptic neurones was microglia-dependent. Since these findings were associated with defects of spatial orientation in recovered wild-type mice, the authors concluded that microglia contribute to pathogenesis. However, the outcome of infection in microglia-depleted Il34LacZ/LacZ mice was not reported (Il34LacZ/LacZ (microglia-depleted) mice  mice . Thus, ied) mice , 74, thied) mice . Microgled) mice . As for ed) mice , 84. KeyAs discussed above, clinical studies have reported associations between inflammatory cytokine production and adverse outcomes. Interestingly, despite the suspicion that microglia are central to pathogenic neuroinflammation, microglial depletion was unexpectedly associated in some studies with an increase in the synthesis of cytokines and/or chemokines in the brain of infected animals –77, whera correlation between increased viral replication and inflammation–noted in one of these studies  and prevthe activation of CNS-resident cell types, such as astrocytes, which was similarly noted in one study , as in othe loss of an immunoregulatory function of microglia, as mentioned above.Overall, further work is needed to clarify the potential relationship between microglial depletion and immune dysregulation in the CNS.hi CD11b+) was reported in some instances  by which microglia (and possibly other CNS resident macrophages) mediate these protective effects . An intrCD: conceptualization and funding acquisition. CH and CD: literature searching, manuscript writing, and figures and tables.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Herpes simplex virus (HSV) is a common pathogen, infecting 85% of adults in the United States. After reaching the nucleus of the long-lived neuron, HSV may enter latency to persist throughout the life span. Re-activation of latent herpesviruses is associated with progressive cognitive impairment and Alzheimer’s disease (AD). As an enveloped DNA virus, HSV exploits cellular membrane systems for its life cycle, and thereby comes in contact with the Rab family of GTPases, master regulators of intracellular membrane dynamics. Knock-down and overexpression of specific Rabs reduce HSV production. Disheveled membrane compartments could lead to AD because membrane sorting and trafficking are crucial for synaptic vesicle formation, neuronal survival signaling and Abeta production. Amyloid precursor protein (APP), a transmembrane glycoprotein, is the parent of Abeta, the major component of senile plaques in AD. Up-regulation of APP expression due to HSV is significant since excess APP interferes with Rab5 endocytic trafficking in neurons. Here, we show that purified PC12-cell endosomes transport both anterograde and retrograde when injected into the squid giant axon at rates similar to isolated HSV. Intracellular HSV co-fractionates with these endosomes, contains APP, Rab5 and TrkA, and displays a second membrane. HSV infected PC12 cells up-regulate APP expression. Whether interference with Rabs has a specific effect on HSV or indirectly affects membrane compartment dynamics co-opted by virus needs further study. Ultimately Rabs, their effectors or their membrane-binding partners may serve as handles to reduce the impact of viral re-activation on cognitive function, or even as more general-purpose anti-microbial therapies. Herpesvirdiae. HSV is prevalent in virtually all populations worldwide, with an estimated 85% infection rate in adults in the United States. HSV primarily enters epithelial cells of mucous membranes and replicates within them, then secondarily enters adjacent sensory nerve termini. Once inside the neuron, HSV travels retrograde within the sensory process to reach the neuronal cell nucleus in the trigeminal ganglion, which lies just outside the brain stem, where virus either replicates or goes latent. Latent viral DNA may re-activate in human, expressing viral proteins to produce new infective virus. Newly produced virions then travel outward by anterograde transport (Herpes simplex virus Type 1 (HSV) is a human enveloped double-stranded DNA virus and member of the alpha herpesvirus subfamily of ransport  from theransport .Herpes simplex virus is an enveloped virus. Thus, most steps in its life cycle depend on membrane trafficking within infected cells. Hence the Rab proteins, small GTPases, which are master regulators of cellular membrane dynamics , are thoNew observations have sparked renewed investigation into the possibility that HSV enters the brain, causing pathology. Two of these are particularly intriguing: (1) HSV interacts dynamically with host cell amyloid precursor protein APP; . APP is n = 14) cohort of autopsied AD and normal brains tested for HSV DNA in three brain regions by PCR  in the brain stem for HSV, which would be a likely portal for HSV entry into the brain from a lip infection , nor theA challenge to correlating anatomical location of HSV DNA with plaques and tangles is the fact that HSV travels long distances inside neuronal projections after replication in the cell body. Hence the location of viral replication, where HSV DNA is most abundant and thus detectable by PCR, may not be the site of plaque pathology, which appears to occur primarily at distant synapses.epsilon4 allele  experiments in HSV infected epithelial cells, accompanied by assessing infectious titers, viral protein expression, and imaging (either fluorescence- or electron-microscopy) .trans-Golgi network and its associated membranes rendered these compartments unable to support secondary envelopment of virions.In an initial experiment 37 different Rab GAPs were over-expressed in three types of human and mammalian epithelial cells . Over-extrans-Golgi network where they may be recruited for secondary envelopment. Trafficking of viral glycoproteins from and to the plasma membrane may be a critical component by which virus escapes immune surveillance  time scales. Disruption of Rabs perturbs the delicate balance of these many moving, fluid compartments. Hence it is no surprise that knockdowns of the management system, the Rabs, would interfere with packaging of enveloped virus, and all other steps in the viral infection and production process. Whether this interference is direct, through interactions with the viral products, or an indirect consequence of cellular membrane trafficking disruption, remains unclear. More precise interventions will be needed to identify a specific role for any of the Rabs in HSV life cycle. Tools for such specific interference could include modified effector molecules that bind to viral proteins and inhibit or promote Rab binding to membranes containing viral products, or drugs that interfere with membrane lipid modifications required for Rab membrane docking. Such experimental manipulations could also have a therapeutic use.Are the Rab GTPases potential therapeutic targets for HSV infections? If, as we suggest here, HSV re-activation is a risk factor for brain pathology such as (but not limited to) AD, then could inhibition of a specific Rab GTPase be a potential therapy? This idea has been proposed previously . HSV is Rabs have also been implicated in cancer progression, with the consequence that targeted Rab inhibitors are under development . The ubiAll datasets generated for this study are included in the manuscript and/or the Supplementary Files.EB developed the ideas, directed the experiments and wrote the manuscript. CW provided the labeled endosomes, added insight to the ideas, performed some of the Western blots, participated in the experiments and interpretation, edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Autophagy (from Greek: ‘self-eating’) is a major catabolic process in eukaryotic cells in which cytoplasmic components are collected and delivered to the lysosomes or vacuoles for recycling. It plays a paramount role in plant fitness and immunity. At present, the frontiers of our understanding of the process are extending exponentially, with new, plant-specific mechanisms and functions being uncovered. In this special issue, original research articles and reviews enlighten this knowledge from lipid metabolism and dynamics, membrane trafficking and proteolysis to pathogen-mediated modulation of the process and the emerging role of autophagy-related approaches in crop improvement.Journal of Experimental Botany contains a collection of reviews and original research papers at the cutting edge of plant autophagy research, and will be valuable not only to ‘autophagists’ but also, considering the truly multifunctional nature of autophagy, all plant scientists.Today, after less than two decades since the first genetic analyses of plant autophagy  and other proteins in directing the autophagy process, the major constituents of the autophagic membranes (i.e. lipids) remain far less well understood. The autophagy process In addition to membranes, lipid droplets are another major pool of lipids in cells. During nutrient deprivation or stress, triacylglycerols (TAGs) stored in these bodies are catabolized into fatty acids that fuel cellular rates of β-oxidation. Lipid droplets are, therefore, crucial for maintaining cellular energy homeostasis and, in animals, autophagy plays a dual role, participating in both their deposition and their degradation  mutant seedlings . Does this lipid have a function in autophagosome membrane formation/structure besides its signalling activity? How is the PI3-kinase complex recruited at the phagophore assembly site in plant cells, which lack Atg14, the autophagy-specific PI3-kinase component?The platform of phagophore assembly. Is the endoplasmic reticulum (ER) the only platform for phagophore assembly in plant cells? Are membrane contact sites involving the ER required for autophagosome formation, similarly to other organisms?The existence of endosomal compartments, such as amphisomes, in which endosomal and autophagic transport merge.Direct participation of autophagy in the deposition and breakdown of lipid stores.The existence of a functional plant analogue of the yeast vacuolar Atg15 lipase.atg mutants, except for atg6, are viable and do not display major developmental defects.The reason why all Arabidopsis The possibility of autophagy in plant cells that do not contain lytic vacuoles (e.g. embryonic cells with storage vacuoles). In addition, if this is the case, what compartment is responsible for degradation of autophagic cargo?The existence of microautophagy (i.e. direct vacuolar engulfment of the cytoplasmic cargo) in plants.The mechanistic role of autophagy in cell death.The composition of the cargo of defence-related selective autophagy. Is the cargo degraded or secreted?Nutrient remobilization from source to sink organs. How and under which conditions is autophagy essential for this process?The sources of nutrients specifically remobilized by autophagy.The extent to which autophagy is critical in cell resource management and degradation, and the balance between cell survival and death.Fitness costs of enhanced autophagy.trans-Golgi network  modification of Atg proteins through limited proteolysis, (ii) crosstalk between the ubiquitin–proteasome system and autophagy pathway, and (iii) digestive proteolysis of autophagic cargo in the lysosomes or lytic vacuoles (for reviews see atg mutants have increased endopeptidase and carboxypeptidase activities (atg plants versus wild-type plants, atg mutants and as a potential compensatory mechanism for protein degradation under autophagy deficiency (atg plants are in agreement with a selective route of autophagy for the disposal of the 26S proteasome, as recently discovered in Arabidopsis (It was previously shown that Arabidopsis In animals, the cysteine cathepsins, which are members of the PLCP family, are major executioners involved in lysosomal protein degradation during autophagy . In planATG5 or ATG7 facilitates Atg8 lipidation, and as a result stimulates both autophagosome formation and autophagic flux.Regulation of autophagy is complex, including transcriptional, post-transcriptional and post-translational steps (ATG8f–GFP reporter lines, the authors demonstrate high autophagic activity in embryos at late developmental stages. Comparison of seed development in atg5 plants versus wild-type plants has revealed that autophagy deficiency compromises storage protein deposition and promotes embryonic arrest and seed abortion.Genetic suppression of autophagy in plants correlates with an overall decrease in plant fitness, including reduced vegetative growth and fecundity, accelerated senescence and enhanced susceptibility to diverse types of stresses. Since autophagy is essential for nitrogen remobilization and seed filling (ATG5 or ATG7 was sufficient to increase autophagic flux in Arabidopsis, Since mere overexpression of ATG5- or ATG7-overexpressing plants was accompanied by increased resistance to oxidative stress and the necrotrophic fungus Alternaria brassicicola (Pseudomonas syringae pv. tomato DC3000 (Plants must cope with growth–defence trade-offs throughout their lives, and maximizing growth-related traits is often achieved at the expense of compromised stress resistance (Autophagy machinery is an integral component of immune systems (Considering the critical role of autophagy in plant fitness and immunity, This is an exciting time for plant autophagy research, and very inviting for new scientists, thus we hope it will continue to bring new discoveries with strong benefits for human health, plant production and the bioeconomy.


Long non-coding RNAs (lncRNA) are emerging as new genetic/epigenetic regulators that can impact almost all physiological functions. Here, we focus on the long non-coding steroid receptor RNA activator (SRA), including new insights into its effects on gene expression, the cell cycle, and differentiation; how these relate to physiology and disease; and the mechanisms underlying these effects. We discuss how SRA acts as an RNA coactivator in nuclear receptor signaling; its effects on steroidogenesis, adipogenesis, and myocyte differentiation; the impact on breast and prostate cancer tumorigenesis; and, finally, its ability to modulate hepatic steatosis through several signaling pathways. Genome-wide analysis reveals that SRA regulates hundreds of target genes in adipocytes and breast cancer cells and binds to thousands of genomic sites in human pluripotent stem cells. Recent studies indicate that SRA acts as a molecular scaffold and forms networks with numerous coregulators and chromatin-modifying regulators in both activating and repressive complexes. We discuss how modifications to SRA's unique stem-loop secondary structure are important for SRA function, and highlight the various SRA isoforms and mutations that have clinical implications. Finally, we discuss the future directions for better understanding the molecular mechanisms of SRA action and how this might lead to new diagnostic and therapeutic approaches. Recent analysis of numerous high-throughput mammalian genomic platforms suggests that protein-coding genes constitute only 2% of the genome, while many non-coding regulatory elements are transcribed into non-coding RNA (ncRNA) –6. IncreSRA1 gene. SRA was initially identified in 1999 by a yeast two-hybrid assay in a human B-lymphocyte library, in which the activation function 1 domain (AF-1) of progesterone receptor (PR) was used as bait (One notable lncRNA is the steroid receptor RNA activator (SRA), which is encoded by the  as bait   of different lengths , and plays an important role in infant development  promoter . These Min vivo, the first mouse model of global SRA gene knockout (SRA−/−) was generated .We have recently revealed that, in mice, SRA is expressed most highly in white and brown adipose tissues compared to other tissues . This unenerated . ConsistWe have described that SRA has diverse physiological functions in regulation of NR signaling, myocyte, and adipocyte differentiation. SRA is also implicated in the pathogenesis of breast and prostate cancers, as we discuss later in this review. Given these diverse functions, it is perhaps not surprising that SRA exerts these effects via various mechanisms and signaling pathways Figures , 3.As described above, SRA regulates NR transcriptional activities through directly or indirectly interacting with NRs Table . SRA alsZhao and colleagues demonstrated that a pseudouridine synthase (PUS), mPus1p, can function as a coactivator for mouse retinoic acid receptor gamma (mRARγ) and other nuclear receptors . They fu. first recognized the role of SRA in regulating cell proliferation via studies of mammary epithelial cells  were also affected by SRA knockdown . InteresThe above transcriptomic analyses illustrate the diverse mechanisms through which SRA's transcriptional regulatory actions might impact cell biology. To further address this, we have recently identified SRA-responsive genes in SRA-overexpressing ST2 adipocytes and SRA knockdown 3T3-L1 adipocytes . In SRA-As described above, in both cancer cells and adipocytes, SRA-responsive genes include those involved in transmembrane receptor-mediated signal transduction, suggesting that SRA may regulate pathways responsive to extracellular effectors Figure . Indeed,Interestingly, a recent study revealed that SRA associates with the RNA helicase Ddx5/p68 in murine Beko pre-T cells, and acts as a coactivator in the Notch signaling pathway  Figure . It remaMost early studies, based on reporter gene assays, revealed that SRA functions as a scaffold to coactivate multiple transcriptional activities of NRs. The molecular mechanisms underlying this coactivator function have begun to be elucidated Figure . Thus, SIGF2 and increased the interaction between the endodermal enhancer and the IGF2 promoter. SRA also interacts with members of the cohesin-binding complex, and knockdown of SRA reduces cohesin binding although this does not affect CTCF genomic-binding sites.Chromatin insulators are DNA elements that can protect a gene from outside influence, leading to either inappropriate activation or silencing of the gene , 80. CTCIncreasing evidences demonstrate that lncRNAs can target diverse chromatin regulators and several chromatin modifications , 16. ModThe need to understand these mechanisms in the context of cancer is particularly important, because there are increasing evidences showing that SRA plays an important role in the development of breast and prostate cancers. However, SRA has also been implicated in the pathogenesis of other diseases Figure . ConsequSRA expression is significantly up-regulated in human tumors of steroid-responsive tissues including the breast, uterus and ovary , 68. OneSRA1 locus . We have found that SRA−/− mice are resistant to high-fat diet (HFD)-induced obesity, with decreased fat mass and increased lean mass  is a major triacylglycerol (TAG) hydrolase that acts as a key regulator of hepatic lipid metabolism. Our recent study demonstrated that loss of SRA in hepatocytes upregulates ATGL expression and the β-oxidation of free fatty acids (FFA), whereas forced expression of SRA inhibits these outcomes  is one of the most common heterogeneous endocrine and metabolic disorders. PCOS accounts for 75% of anovulatory infertility and is highly associated with obesity, insulin resistance, dyslipidemia and increased risk of diabetes and cardiovascular events . A recenOther recent case studies have identified three independent families with inactivating SRA1 mutant variants  that are associated with delayed puberty/normosmic idiopathic hypogonadotroic hypogonadism (IHH) . This diIn addition to PCOS and IHH, two final studies implicate SRA in the pathogenesis of endometriosis, an estrogen-dependent disease that affects about 10% of reproductive-aged women. Interestingly, Lin and colleagues reported that SRA RNA and ERα protein expression are higher in endometrial tissue compared to ovarian tissue surrounding mature teratoma and ovarian endometrioma . In contIn summary, studies relating to the pathogenic roles of SRA have focused largely on breast and prostate cancers, as well as metabolic diseases such as obesity-related insulin resistance and hepatic steatosis. Other studies are beginning to highlight potential roles of SRA in other diseases, including cardiovascular disease and reproductive disorders such as PCOS, IHH, and endometriosis. Future studies must now build on these promising findings, in particular through human analyses, so that we can better understand the pathogenic roles of SRA.It has been almost two decades since the discovery of the lncRNA SRA. However, many aspects of SRA biology remain to be elucidated. For example, what is its exact physiological function? Through what molecular mechanisms does SRA regulate gene expression, development, and cell differentiation? Finally, how does SRA impact the pathogenesis of cancers and other diseases?In terms of molecular mechanisms, we now know that SRA exists in the ribonucleoprotein complex that includes SRC-1 and binds to components of proteins belonging to both the TrxG and PRC2 complexes, which regulate chromatin's epigenetic state. SRA also binds to numerous protein partners including NRs and other transcription factors and coregulators. Thus, it is crucial to further identify the dynamic SRA-binding proteins or RNAs in living cells, including how these interactions are influenced by hormones or other physiological conditions. ChIRP is one approach that has much promise in revealing this new knowledge. In addition, how SRA-binding sites are regulated by different extracellular signals, in a cell-context dependent manner, is largely unknown. Therefore, it is necessary to identify SRA genome-wide binding sites and analyze their association with gene expression under specific differential conditions, developmental stages and in different cell types, such as during adipogenesis or breast cancer cell oncogenesis. This knowledge would improve our understanding of SRA's transcriptional targets and how these impact physiological and pathological processes.A related question is, how is SRA recruited to chromatin? Clearly, this recruitment may be via known SRA-interacting partners, including NRs, other transcription factors (such as MyoD), coregualators (p68), and components of chromatin regulatory complexes . However, other lncRNAs employ more diverse mechanisms, including the formation of RNA:DNA:DNA triplexes; generation of RNA:DNA hybrids that displace a single strand of DNA; or creation of an RNA:RNA hybrid between a lncRNA and a nascent transcript , 16. DoeIncreasing evidences indicate that SRA can both activate and repress gene expression Figure . AlthougWe and others have shown that SRA regulates insulin, TNFα and Notch signaling pathways, but many questions remain. For example, what is the molecular mechanism underlying this regulation? What are the upstream direct targets  that regulate SRA function in response to these signaling pathways?−/− mice lose both SRA and SRAP in all tissues  occurs in other developmental or pathological contexts. For example, is the intron-1-containing SRA also enriched in the adipose tissue or liver of obese individuals, compared to healthy non-obese controls? These splicing processes are regulated by trans-acting proteins (repressors or activators) and corresponding cis-acting sites in the DNA promoter sequence of the nditions , 104 is LncRNAs have been emerging as factors that provide a new layer for regulation of gene expression in numerous biological contexts, including cell differentiation, development, physiology, and disease. In addition to being a potential biomarker for breast cancer, the differential expression of SRA and genetic mutations have been connected to other diseases such as POCS, IHH, endometriosis, and cardiovascular dysregulation. However, only a handful of studies have reported a role for SRA in these contexts, underscoring the need for additional research.Experimental therapeutic agents targeting lncRNAs are only in their infancy. The characterization of the precise secondary structures of SRA promises to open new strategies. For example, it might be possible to target specific stem loop structures of SRA to more precisely modulate activities of nuclear receptors  and otheMuch progress has been made in understanding the physiological and pathological functions of SRA, and some light has been shed on the underlying molecular mechanisms. However, from this review it is clear that many, many questions remain to be answered. Nevertheless, SRA and other lncRNAs display promise as biomarkers and therapeutic targets that might allow new strategies for improved clinical outcomes. To realize this promise, it is essential that future research yields further insights into the physiological and pathological roles of SRA, as well as the molecular mechanisms of its action.LS, LY, DZ, and BX wrote the manuscript, and BX designed figures. WC and BX revised the manuscript, and WC rewrote several sections of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.