J. Clin. Invest. 132, e148554]. Activated fibroblasts are critical regulators of cardiac wound healing after injury, but their excessive and persistent activation promote tissue fibrosis, a hallmark feature of the pathological remodeling of the heart. While much of the previous work in cardiac fibroblast biology has focused on the role of canonical signaling pathways or components of the extracellular matrix, recent efforts have been focused on deciphering the gene regulatory principles governing fibroblast activation. A better understanding of the molecular mechanisms that trigger and sustain the fibrotic process in heart disease has the potential to accelerate the development of therapies that specifically target the cardiac activated fibroblasts, which are at the moment unavailable. This concise review focuses on the mechanisms underlying the chromatin and transcriptional regulation of cardiac fibroblast activation. We discuss recent work from our group and others in this space, highlighting the application of single-cell genomics in the characterization of fibroblast function and diversity, and provide an overview on the prospects of targeting cardiac fibroblasts in heart disease and the associated challenges.Cardiac fibroblasts play an essential role in maintaining the structural framework of the heart. Upon stress, fibroblasts undergo a cell state transition to activated fibroblasts , a highly synthetic cell type that proliferates, migrates, and secrets both extracellular matrix as well as signaling factors that can modulate cellular crosstalk [ These limitations have been circumvented following the generation of powerful new mouse models [in vivo, providing cogent evidence that the activated fibroblast is a major driver of disease pathogenesis.Fibrosis is a stereotypical wound-healing response that is mounted after tissue injury or stress and is often associated with organ dysfunction in a variety of human diseases. In the context of heart disease, the leading cause of mortality in the developed world, a significant body of clinical data has demonstrated that cardiac fibrosis is strongly associated with adverse outcomes in several conditions, including heart failure with reduced ejection function (HFrEF), heart failure with preserved ejection function (HFpEF), and genetically driven cardiomyopathies . While te models , which he models  or the de models  , confirming that TGF-β signaling is a nodal regulator of profibrotic function in the heart [Smad3 deletion is also associated with detrimental effects in cardiac function [Several signaling pathways have been implicated in the regulation of cardiac fibroblast activation and fibrosis. Among these, the canonical and noncanonical TGF-β signaling, the adrenergic receptor and the renin-angiotensin-aldosterone systems (reviewed extensively elsewhere ). Withinposition . Several failure , 8. Morehe heart . Of notefunction , highligfunction . Within function . Furtherfunction . Howeverin vivo remains an unanswered question with important therapeutic implications.The stress-induced pathways that trigger fibroblast activation ultimately converge on transcription factors (TFs) and the chromatin regulatory apparatus in the nucleus, which transduce these broad upstream signals into changes in gene expression and cell identity. Fibroblast activation, like any other cell state transition, is initiated by the binding of TFs and chromatin regulators at the level of the DNA, which triggers the activation and repression of a defined set of enhancers. Among the epigenetic modifications, acetylation of lysine residues (Kac) on histone tails is a major feature of chromatin activation. Importantly, previous studies have demonstrated a critical role of histone acetyltransferases and histone deacetylases in controlling cardiac profibrotic function (extensively reviewed elsewhere ). Contexin vivo, coupled with single-cell genomics, offers the unique opportunity to interrogate which cell types, gene programs and molecular mechanisms are dynamically regulated in disease pathogenesis. To explore these approaches in the context of the TAC model of pressure-overload heart failure, we have recently combined the use of single-cell RNA- and ATAC-sequencing  in the setting of intermittent BET bromodomain inhibitor exposure [The use of small molecule compounds that reversibly inhibit stress-dependent transcription exposure . In thisexposure . Our scRMeox1, a poorly studied TF known to control sclerotome polarization during development [Meox1 was also dynamically regulated in the setting of stress and exposure to JQ1: strongly increased in fibroblasts upon TAC, abolished by JQ1 treatment, and robustly induced again upon JQ1 withdrawal [Meox1 is characterized by multiple distal accessible regions, with several of these being sensitive to TAC and BET inhibition. As enhancers are pervasively transcribed and nascent transcription is a robust indicator of enhancer activity [in vitro to map genome-wide RNA polymerase II nascent transcription and identify putative active enhancers. This highlighted that within the large regulatory element proximal to Meox1, one specific region (which we named Peak9/10 enhancer) was associated with a strong increase in nascent transcription following TGF-β-induced fibroblast activation in vitro. We used CRISPR interference to repress a series of distal elements within the Meox1 regulatory region and found that the Peak9/10 enhancer was specifically required for Meox1 transactivation upon TGF-β stimulation, while other accessible regions identified in vivo were not [One of the enhancer elements whose chromatin accessibility was most anticorrelated with left ventricular systolic function was a large enhancer proximal to elopment . The expthdrawal . The laractivity , we perfwere not . This hiMeox1 as a putative regulator of stress-dependent fibroblast activation, we performed a series of in vitro assays that demonstrated how Meox1 depletion in TGF-β treated cells decreased the formation of smooth muscle actin (SMA)-positive stress fibers, collagen-gel contraction and EdU incorporation, suggesting that MEOX1 was required for contractile and proliferative phenotypic transitions in vitro, two functional hallmarks of stress-activated fibroblasts. Finally, we combined MEOX1 ChIPseq with PROseq in the context of Meox1 depletion to demonstrate that upon fibroblast activation, MEOX1 binds and directly regulates the transcription of a specific set of stress-responsive distal and gene elements. These findings suggest MEOX1 functions as a transcriptional mediator of the cell state transition between quiescent and activated fibroblast in vitro. Future studies are needed to explore the role of MEOX1 in vivo to understand if this TF is a critical regulator of fibroblast activation and fibrosis in heart failure pathogenesis. Of note, recent single-cell studies exploring the transcriptional landscape of the human heart in healthy and diseased tissues highlighted how MEOX1 is expressed in human cardiac fibroblasts and up-regulated during disease progression [in vivo. Though, TFs have historically been considered undruggable due to their structure and complex functions. Within this context, there has been particular excitement around the possibility of using proteolysis targeting chimeras (PROTACs) [To understand the role of gression , 21. It PROTACs)  to targePOSTN and FAP as well as previously unreported genes such as NALF1 and TSHZ2. An elegant in vitro CRISPR interference assay coupled with immunofluorescence to measure changes in SMA content revealed how the depletion of a subset of these genes decreased profibrotic response. Another recent study has utilized snRNA-seq on cardiac specimens obtained from human donors affected by different types of congenital heart disease (CHD), including hypoplastic left heart syndrome (HLHS) and Tetralogy of Fallot (TOF) [POSTN, FAP and MEOX1.Single-cell genomics offers the unprecedented opportunity to unbiasedly capture the transcriptional landscape of complex tissues such as the heart. Several studies have recently used single-cell approaches to investigate fibroblast transcriptional heterogeneity in cardiac pathologies. Single-nucleus RNA-sequencing (snRNA-seq) on cardiac biopsies from controls and patients with dilated cardiomyopathy (DCM) or hypertrophic cardiomyopathy (HCM) has highlighted how, amongst all cellular compartments, the fibroblasts were the most transcriptionally dysregulated in patients with cardiomyopathy . Indeed,ot (TOF) . Again, Pdgfra positive cells at days 3 and 7 post myocardial infarction (MI) identified novel fibroblast cell states, including a population characterized by the expression of canonical WNT pathway inhibitors along with several stress-activated fibroblasts subtypes with pro- or anti-fibrotic signatures [Wt1 to sort epicardial-derived cells and performed scRNA-seq at several time-points  post-MI in two inbred strains, C57BL/6J and 129S1/SvImJ [Several studies have also sought to characterize fibroblast transcriptional heterogeneity and dynamics in murine models of heart failure. ScRNA-seq in non-myocyte gnatures . SimilarS1/SvImJ . This daCilp and Thbs4, that do not correspond to the classical SMA–expressing activated fibroblasts. Notably, another group has recently used scRNA-seq in the TAC model of pressure overload and identified a cardiac fibroblast subpopulation which expands upon injury that acquires Thbs4 expression [Another study dissected the fibroblast transcriptional changes in response to angiotensin II (AngII) infusion, another established murine model of cardiac hypertrophy . Interespression . While tFibroblast biology is complex and fascinating as these cells must maintain tissue architecture and participate in organ homeostasis across diverse organ systems. Fibroblasts are critical regulators of repair and wound healing, but their chronic activation results in tissue fibrosis, a finding that often correlates with organ dysfunction. Importantly, fibrosis is a shared feature across a variety of cardiac pathologies ranging from ischemic to inflammatory heart disease and represents a final common pathway in the development of heart failure regardless of the underlying etiology. Furthermore, the presence of patchy scar within the myocardium further predisposes patients to additional comorbid conditions including ventricular arrhythmias and sudden cardiac death. Given the central role of fibrosis across these diverse conditions, it is appealing to speculate that the development of anti-fibrotic therapies has the potential to benefit a broad swath of patients suffering from cardiac disease. While the notion of decreasing or depleting activated fibroblasts may represent a rational therapeutic strategy for treating a variety of heart disease , many chTbx20 in the heart [Finding tissue and organ specific fibroblast features has historically been a significant challenge. As these cells reside across tissues with varied and distinct functions, it is likely that their ability to accomplish diverse tasks is, in part, due to defined tissue-specific gene regulatory networks. Consistent with the fact that organ fibroblasts are not generated from a common progenitor pool but instead arise independently during organogenesis, a recent single-cell study showed that organ fibroblasts possess defined molecular blueprints characterized by the expression of developmental TFs, such as he heart . Other she heart . These fcis regulatory elements possess increased tissue-specific activity as compared with protein coding genes [Given the challenges associated with identifying tissue-specific fibroblasts markers, it is possible to speculate that the non-coding genome could provide more tissue-restricted targets that can be explored for therapeutic manipulation. Indeed, ng genes  and are ng genes . Apart fin vivo, their use is associated with on-target toxicity in off-target tissues due to the broad expression of BET proteins. Further mechanistic refinement of the modalities by which downstream BET targets contribute to fibroblast activation will offer opportunities to develop therapeutic approaches that are tailored to targeted gene regulation specifically in the fibroblast cell compartment. In parallel, studies exploring the use of more specific BET inhibitors , the first functionally relevant cardiac fibroblast-specific long non-coding RNA [Wisper was up-regulated following MI in mice, has a human ortholog and its depletion in vivo using an antisense oligonucleotide  was associated with marked reduction in cardiac fibrosis and a significant improvement in left ventricular systolic function. Importantly, Wisper expression was not detected in lung or kidney fibroblasts, supporting the notion that Wisper may have specificity for mediating cardiac fibrosis. Indeed, the ASO field is an emerging area of drug development that offers a promising alternative to targeting enhancers implicated in fibroblast activation. Targeting transcripts such Wisper that possess tissue- and cell-specificity could represent a valid approach to therapeutically target stress-activated cardiac fibroblasts with systemic delivery of nucleic acid-based therapies to extrahepatic tissues remaining one of the greatest barriers. The exquisite specificity of enhancer activity, when combined with their role as highly specialized nodal regulators of gene expression, make these genomic elements an incredibly attractive therapeutic target for heart disease.An enormous challenge in the translational potential for enhancer research lies in how to therapeutically target these regions. We and others have used BET inhibitors to target fibroblast enhancers and inhibit gene expression , 31. Altmodomain , 33) in modomain , 35. A smodomain , 37. We ding RNA . Wisper The concept that aberrant cell states can be targeted for ablation is well-established in cancer biology. Chimeric antigen receptor (CAR) T-cell therapy is a new form of immunotherapy that utilizes specially altered T-cells to target and eliminate specific cancer cells. In recent years, there has been growing interest in leveraging this therapeutic concept to non-oncology settings, including heart disease. Recent work from Jonathan Epstein's laboratory has been pivotal in establishing the proof-of-concept that CAR T-cell therapy can be used to target cardiac fibroblasts to reduce fibrosis and improve heart function in murine models of heart failure . These ain vivo [The homeostasis of the heart, or that of any organ, relies on a complex crosstalk between different cell types with specialized functions. Recent advances in single-cell ‘omics’ approaches have highlighted the heterogeneity of the cellular composition of the heart, revealing multiple subsets of cardiomyocytes, fibroblasts, endothelial and immune cells with diverse developmental origins and molecular properties . Sophistin vivo . FactorsOur current understanding of how other cardiac cell compartments influence fibroblast cell states via cell-to-cell communication is limited. This is particularly important as we envision therapies that aim to substantially improve heart disease by targeting activated fibroblasts. Even if we would be able to safely and effectively decrease or eliminate activated fibroblasts, would this be sufficient to treat chronic heart disease? If another cellular compartment that provides a critical upstream signal for fibroblast activation remains unaffected, will the remaining quiescent fibroblasts keep transitioning to an activated state in the context of this chronic stress? Here again, single cell approaches will be pivotal in helping to identify specific cell compartments that can engage in direct communication with fibroblasts to affect heart disease outcomes. A better understanding of the molecular mechanisms driving cellular crosstalk will aid in designing therapies that most effectively target fibroblast activation in heart disease.It is becoming increasingly clear that cancer-associated fibroblasts (CAFs) are a key component of the tumor microenvironment with many diverse functions, including remodeling of the ECM that can in turn modulate tumor stiffness and facilitate tumor progression, mediating reciprocal signaling interactions with cancer cells, and facilitating crosstalk with immune cells , 44. As Due to the lack of therapies that directly target excessive fibroblast activation, the global health burden of fibrotic disorders including cardiac fibrosis is strikingly high .Ongoing efforts to uncover the complex transcriptional and epigenomic regulation of cardiac fibroblasts activation with technologies such as single-cell genomics are increasing our understanding of the mechanistic basis of fibroblast activation.Such studies may ultimately inform novel therapeutic targets and strategies to treat various forms of heart disease that feature maladaptive remodeling of the fibroblast states and tissue fibrosis.


Myocardial injury often leads to heart failure due to the loss and insufficient regeneration of resident cardiomyocytes. The low regenerative potential of the mammalian heart is one of the main drivers of heart failure progression, especially after myocardial infarction accompanied by large contractile muscle loss. Preclinical therapies for cardiac regeneration are promising, but clinically still missing. Mammalian models represent an excellent translational in vivo platform to test drugs and treatments for the promotion of cardiac regeneration. Particularly, short-lived mice offer the possibility to monitor the outcome of such treatments throughout the life span. Importantly, there is a short period of time in newborn mice in which the heart retains full regenerative capacity after cardiac injury, which potentially also holds true for the neonatal human heart. Thus, in vivo neonatal mouse models of cardiac injury are crucial to gain insights into the molecular mechanisms underlying the cardiac regenerative processes and to devise novel therapeutic strategies for the treatment of diseased adult hearts. Here, we provide an overview of the established injury models to study cardiac regeneration. We summarize pioneering studies that demonstrate the potential of using neonatal cardiac injury models to identify factors that may stimulate heart regeneration by inducing endogenous cardiomyocyte proliferation in the adult heart. To conclude, we briefly summarize studies in large animal models and the insights gained in humans, which may pave the way toward the development of novel approaches in regenerative medicine. Cardiovascular diseases (CDVs) are a group of the most common, serious, chronic, and life-threatening disorders causing death, disabilities and huge socioeconomic costs globally. A common characteristic of most non-congenital heart diseases is the extensive loss of contractile cardiomyocytes. For example, severe MI can wipe out more than 25% of ventricular cardiomyocytes within a few hours . SubsequCurrent strategies to replace lost cardiomyocytes rely on either the stimulation of endogenous regenerative processes, including the stimulation of cardiomyocyte proliferation and the conversion of other cardiac cell types,  , or the Planarians, Annelids and Hydra represent the group of invertebrates having a spectacular capacity to regenerate all tissues and organs by both tissue remodeling and through the proliferation of pre-existing and resident adult somatic stem cells. Additionally, some lower vertebrates such as newts, axolotls and teleost fish can regenerate fins, limbs and tails after traumatic damage [The first evidence of complete regeneration after amputation is seen in lower clades of the animal kingdom. c damage , 71. TheDanio rerio) can undergo complete cardiac regeneration without scar formation within 2 months after 20% apical resection of the ventricle through a robust proliferation of resident cardiomyocytes [blastema) responds to the stimulus of an amputation. Differentiation of these cells results in new tissues functionally integrated with the pre-existing tissue [Recently, studies have demonstrated that amphibians and zebrafish also retain the ability to regenerate the heart even as adults. Notably, zebrafish  generation [2 is obtained and then maintained for 2 weeks). Strikingly, induction of MI under these conditions resulted in a robust regenerative response with decreased myocardial fibrosis and improved left ventricular systolic functions [2 concentration), the development of postnatal endothermy and the associated increase in thyroid hormones have also been proposed as a brake for the cardiac regenerative potential in mammals [Similar to zebrafish, embryonic and neonatal murine cardiomyocytes require lower oxygen for metabolic processes using glycolysis as a source of energy. Indeed, it is well documented that for a few days, the neonatal mouse heart retains full regenerative potential as demonstrated in the apical heart resection and left anterior descending artery occlusion myocardial infarct (MI) mouse models , 76. Impneration , which pneration . In suppunctions . In addi mammals , 70.Interestingly, the hypothesis that mammalian hearts might have regenerative potential dates back to 1956. In this pioneering study, investigators are able to show how the healing processes in young rats, subjected to a myocardial lesion, proceed rapidly due to high regenerative abilities . HoweverTaken together, these observations confirm that cardiac regeneration in mammals per se is possible. However, it is extremely limited and blunted immediately after birth, but the intriguing possibility to extend regenerative responses to adulthood holds potential for the identification of regenerative treatments for heart failure. In this context, the establishment of in vivo models of complete cardiac regeneration are instrumental for dissecting molecular networks and providing a platform to test novel targeted therapies for the stimulation of cardiomyocyte proliferation and cardiac regeneration in mammals. In the following pages, we provide an overview of the currently available animal models employed to study cardiac regeneration and furthermore summarize their contributions in the identification of molecular and therapeutic approaches for cardiac repair through re-activation of regenerative processes.In the last decade, scientists focused on determining a highly valid and clinically relevant animal model to study heart regeneration. One major challenge is that the heart is a complex organ that requires the concerted action and interaction of different cell types  to mediate the response to distinct types of myocardial damage . The stuThe knowledge of endogenous mechanisms that might regulate the repair and healing of an injured heart in neonatal mice represents a fundamental perspective to stimulate heart regeneration in adult mammals using innate mechanisms. However, a standardized model and a consensus on a method to investigate cardiac regeneration is still lacking. Indeed, three different, challenging, neonatal cardiac injury models are commonly used to investigate cardiac regeneration: the cryoinjury model, the apical resection model and the left anterior descending artery (LAD) ligation model Fig. .Fig. 1NeAll current experimental injury models are performed under hypothermic anesthesia, which is induced by incubating the neonates in ice water for 2–3 min. Generally, to avoid direct contact with ice, a small barrier is created using a sterile gauze . SuccessA series of studies on cardiac regeneration have been conducted using a neonatal mouse model of cardiac cryoinjury. First, a transverse skin incision is carried out across the chest, followed by a thoracotomy procedure. Once the heart is exposed, a liquid nitrogen cooled cryoprobe  is applied to the left ventricular surface for a defined period of time. Next, the chest wall is carefully closed by surgical sutures between the ribs of the opened intercostal space and then the cut pectoral muscles are sutured. Lastly, the skin incision is closed with two sutures . Of noteImportantly, Darehzereshki and colleagues have shown that the type and severity of the cryoinjury strongly affect the regenerative responses in neonatal mice. They have developed different cardiac cryoinjury models to induce different degrees of damage, either inducing severe damage by affecting the full wall diameter , or inflicting mild damage, by not penetrating the entire ventricle wall . A non-transmural cryoinjury in P1 mice can undergo healing without scarring, while hearts with a transmural cryoinjury fail to regenerate. In contrast to other studies employing this model, no statistically significant increase in cardiomyocyte proliferation in either the border or remote areas after transmural or non-transmural cryoinjuries was observed . It is cIn 2014, Mahmoud and colleagues published a Nature Protocol outlining the apical resection method, in which they described a 10–15 min surgical procedure step by step in neonatal mice at P1 or P7. Following hypothermic anesthesia, a thoracotomy was performed across the lower half of the chest. Once the left ventricular chamber (LV) is exposed, the smallest amount of tissue possible should be removed from the apex of the heart . This reIn 2011, the pioneers of this method, Porrello and colleagues, have revealed that the surgical resection of 15% of the left ventricular apex in P1 mice is fully restored within 21 days. Immediately after the resection at P1, a robust inflammatory response occurs and the resection site is sealed by a large blood clot. Gradually, the apical clot is resorbed and replaced by normal myocardial tissue. Within 21 days, proliferating cardiomyocytes have substituted the resected tissue and normal cardiac function is restored . The regBy contrast, results from Andersen and colleagues are contradictory to those reported by Porrello. In fact, they have claimed that apical resection in P1 mice results in irreversible fibrosis and dilated cardiomyopathy suggesting that regenerative cardiac abilities in mammals are limited, even in the early life stages . They haLeft anterior descending coronary artery (LAD) ligation to induce MI in neonatal mice appears to be the most relevant model since it more closely resembles the clinical situation after MI . The patMoreover, several critical points can influence the rate of mortality in neonatal mice throughout the surgery and immediately following the operation. Crucial precautions are already required during the anesthesia stage, including the use of melted ice (ice water) to shorten the hypothermia induction time. Moreover, the overall time that the mice are subjected to hypothermic anesthesia induction should not exceed 2–5 min . An addiNevertheless, from a clinical angle, this procedure remains the most relevant for studying pathobiological and regenerative processes. Several studies using this LAD coronary ligation model confirmed the cardiac regenerative potential after MI at P1 through compensatory cardiomyocyte proliferation , 77. In Notably, the pressure overload model by pulmonary artery banding (PAB) surgery is a recent addition for studying heart regeneration in the neonatal rat. Investigators suggest that this model is quite exhaustive to investigate cardiomyocyte proliferation. Differentially expressed genes between cardiomyocytes from PAB and sham-operated rats are mainly implicated in mitosis and cell division. Additionally after PAB, stress may extend the period of cardiomyocyte proliferation beyond 7 days after birth. The rate of cardiomyocyte proliferation is increased in rats after PAB surgery, concluding that the pressure overload can induce the heart to proliferate at the neonatal stages. Thus, this model may be useful to study congenital heart diseases, such as pulmonary stenosis, tetralogy of Fallot and other chronic heart failure diseases .Similarly, Mahammadi et al. have established a transverse aortic constriction protocol in neonatal mice (nTAC) in the regenerative (at P1) and non-regenerative stages (at P7). The TAC model is commonly used in adult mice to investigate cardiac pressure overload-induced left ventricular heart failure. However, Mahammadi and colleagues aimed to compare potential regenerative responses of neonatal mice to nTAC surgery in P1 versus P7. Their results revealed that nTAC at P1 induces an adaptive response, preserving cardiac function and increasing cardiomyocyte proliferation within the regenerative window phase. In contrast, nTAC in P7 mice, in which the cardiomyocyte proliferation capacity is lost, causes cardiomyocyte hypertrophy, fibrosis and cardiac dysfunction. The authors suggest that the adaptive regenerative response toward pathological pressure overload triggers cardiomyocyte proliferation immediately after birth, thus mimicking physiological hypertrophy that occurs during pregnancy or during sport. In contrast to preserved cardiac function, nTAC outside of the regenerative window is accompanied by cardiac dysfunction and fibrosis, which is typical for pathological hypertrophy as observed in the adult heart exposed to chronic pressure overload , 67.Extra care and exceptional technique are required in the performance of both PAB  and TAC The common concept of the described cardiac regeneration models is to identify molecular switches that promote cell cycle re-entry and mitosis of resident cardiomyocytes, which upon injury can proliferate and replace damaged tissue to maintain cardiac functions. In this section, we highlight recent studies utilizing neonatal cardiac regeneration models to identify factors that could be successfully exploited to induce cardiac regeneration in adult cardiac injury models. Our purpose is to provide a general overview of potential therapeutic targets for enhancing endogenous cardiomyocyte proliferation that may have the potential for translation into larger animal models and potentially humans  in cardiac regeneration. Early administration of recombinant NRG1 prevents transmural scar formation leading to a better outcome of cardiac functions and stimulates cardiomyocytes cell cycle re-entry and proliferation in the first 10 days of life . To deteAnother study has demonstrated the critical role of Meis1 as a master transcriptional regulator of the cardiomyocyte cell cycle. Cardiac-specific Meis1 overexpression inhibits neonatal heart regeneration following MI at P1 via the induction of the cyclin-dependent kinase (CDK) inhibitors p15, p16, and p21. In contrast, cardiomyocyte-specific inactivation of Meis1 in adult mice by using tamoxifen-inducible knock-out mice (αMHC-MerCreMer mice) stimulates cardiomyocyte cell cycle re-entry and re-activation of mitosis .Inflammatory responses and the involvement of bone marrow-derived and cardiac resident immune cells have emerged as crucial factors in the healing of the heart following MI , 30, 84.Importantly, Bassat and colleagues highlighted a critical role of the extracellular matrix for cardiac regeneration. Agrin, an endothelial-derived extracellular matrix component highly expressed at birth, is required for regeneration in neonatal hearts. Treatment of adult mice with recombinant agrin promotes cardiac regeneration after an MI. Mechanistically, the binding of agrin to its receptor, Dag1, induces myofibril disassembly and activation of Yes-associated protein (YAP), a well-described inducer of cardiomyocyte proliferation  , have emerged as master regulators of many biological processes and have already been explored in the field of regenerative medicine.To validate their role, several of these targets have been tested in vitro and in neonatal and adult mouse models in vivo. A few of these targets were also evaluated in large animal models. Mostly, these molecular factors act by stimulating regenerative processes and cardiomyocyte proliferation, but others can promote heart healing by exercising their role on other processes .MicroRNAs are a class of small regulatory molecules with a short size of 20–23 nucleotides. MicroRNAs mainly interact with the 3´ untranslated region (3´ UTR) of target messenger RNAs (mRNAs) through complementary base pairing; and very seldom are also interacting with the 5` UTR or coding gene sequences . Their bHere, we focused on microRNA-based therapies, which stimulate cardiomyocyte proliferation after cardiac injury in mice and result in promising preclinical studies. The first evidence for miRNAs playing a role in cardiomyocyte proliferation was provided by Eulalio et al. Their functional high-throughput screening for 875 human microRNAs in neonatal rat cardiomyocytes identified 204 microRNAs that significantly increased cardiomyocyte proliferation. Two of the most potent miRNAs regarding the induction of cardiomyocyte proliferation in vitro (miR-590 and miR-199a) were selected and further tested in vivo in mice. Neonatal mice are injected with AAV9 virus particles, overexpressing miR-590 or miR-199a, both resulting in an increased number of dividing cardiomyocytes. Infarcted hearts of adult mice (8–12 weeks), injected at the time of LAD ligation with AAV9 vectors expressing the two miRNAs, showed noticeable cardiac regeneration and almost complete recovery of cardiac functional parameters. A search for the relevant targets of these two miRNAs, revealed genes primarily belonging to the cell cycle, cell growth, and proliferation pathways  . FurtherLikewise, a group of miRNAs, miR-17-92, have been investigated for their critical role in postnatal cardiomyocyte mitotic arrest. Deletion of the miR-17-92 cluster in neonatal mice caused a decrease in cardiomyocyte proliferation, whereas overexpression in cardiac-specific transgenic mice (3-week-old) induced cardiomyocytes proliferation. In a disease setting, the effect of miR-17-92 overexpression in tamoxifen-inducible adult mice (4-month-old) subjected to MI by LAD ligation resulted in preserved cardiac functions accompanying an induction of cardiomyocyte proliferation. Mechanistically, miR-17-92 targets PTEN which in turn mediates the regulation of cardiomyocyte proliferation . Later, Porrello and colleagues have elucidated the role of the miR-15 family in repressing cardiac regenerative capacity in mammals after birth. Multiple members of the miR-15 family are up-regulated in mice after birth. MiR-195 (a member of the miR-15 family) transgenic mice subjected to LAD ligation at P1 showed extensive myocardial necrosis and inhibition of cardiomyocyte proliferation. Administration of LNA (locked nucleic acid) modified miR-15 anti-miRs to P1, 7 and 14 mice, followed by LAD ligation at P21 indeed stimulated cardiomyocyte proliferation and promoted myocardial regeneration in adulthood , 77. YanSeveral miRNAs are involved in the withdrawal from the cardiomyocyte cell cycle and in the regulation of cardiomyocyte proliferation. Here, we have summarized the most common miRNAs studied for cardiac regeneration . Therefore, major investigations in vivo have been conducted in rodents, which more closely resemble a complete disease model than an individual cell type, but can still maintain a variable comparability to the exact manifestations of complex human diseases. To face these challenges, the findings from studies in murine models need to be projected to large mammal models which are anatomically and physiologically more similar to humans.In their study, Kim and colleagues have shown that the timing of regenerative arrest is linked to the cell cycle exit and polynucleation of cardiomyocytes. Their studies in preterm (~ 3 weeks before birth) piglets underline that ~ 10% of cardiomyocytes are binucleated, smaller, proliferative with only a few undergoing apoptosis, while on the contrary, in term (2 day before birth) piglets, the rate of binucleated cardiomyocytes increases to ~ 30% . More reALCAPA syndrome  [Whether the human heart possesses a similar regenerative capacity as mice or pigs remains unknown. A recent review highlighted a very early study of Wesselhoeft et al. with congenital pediatric heart disease that causes myocardial ischemia, named  artery) . They ha artery) . Further artery) . These r artery) . Further artery) . These sPresently, several strategies for regenerating the injured and diseased heart are pursued. In addition to stimulating endogenous cardiomyocyte proliferation, transdifferentiation of non-cardiomyocytes, e.g., cardiac fibroblasts that constitute the fibrotic infarct scar, into cardiomyocytes is a possibility that is currently being explored . HoweverTo conclude, despite the numerous insights into cardiac regeneration using fascinating animal models, the regenerative potential of the heart needs further elucidation and tremendous efforts are still required to develop innovative and safe therapies to be translated from experiments to treatments for patients with cardiac diseases.


Atypical femoral fracture (AFF) is a rare complication in patients with osteoporosis undergoing long-term bisphosphonate therapy. The most common symptom of incomplete AFF is nonspecific thigh pain, which is often difficult to distinguish from other causes.We present two cases of AFF misdiagnosed as lumbar radiculopathy. Both patients visited our hospital for thigh pain, and in both cases the findings of lumbar spine magnetic resonance imaging showed substantial nerve compression. These patients had been treated for lumbar radiculopathy, but localized symptoms became conspicuous and femoral radiographs revealed complete AFF. The initial radiographs were reviewed retrospectively and revealed slight lateral cortical thickening in the affected femur, denoting a missed incomplete AFF. Internal fixation with intramedullary nails was performed.AFF may mimic lumbar radiculopathy. Therefore, clinicians should consider AFF as a differential diagnosis in older patients with lumbar canal stenosis who are undergoing long-term bisphosphonate therapy and present with thigh pain. Bisphosphonates are the most commonly prescribed medications for osteoporosis that prevent osteoporotic fractures . SeveralThe mean age of patients with surgically-treated, bisphosphonate-related AFF is reportedly 70 years, with groin and thigh pain being the most common symptoms . FurtherTherefore, the epidemiologic features and symptoms of AFF and lumbar radiculopathy are similar and these two conditions may coexist. However, missed incomplete or nondisplaced AFFs are at high risk of further displacement and may negatively impact the hospital stay, systemic or local complications, and/or mortality . Thus, aA 67-year-old woman visited our hospital with a chief concern of left anterior thigh pain for 1 month without any specific history of trauma. The patient had a history of systemic lupus erythematosus (SLE) and osteoporosis, for which she had been administered a proton pump inhibitor (PPI), 10 mg prednisolone, and monthly alendronate for 5 years.The patient had long-term sensory disturbance in the lower extremities owing to the peripheral neuropathy associated with SLE. The patient reported no rest pain in the left lower extremity, but reported pain during weight-bearing and walking. Further, mild weakness of the iliopsoas and quadriceps muscles was observed, probably because of pain and long-term disuse.Metabolic studies and tests yielded normal results except for 25-dihydroxyvitamin D . Her initial radiographical examination included a lumbar spine series and antero-posterior (AP) pelvis and revealed degenerative changes with reduced disc height at L1-2 . The initial radiographical examination included a lumbar spine series and revealed decreased disc height and grade 2 spondylolisthesis at L4-5 (Fig. An 83-year-old woman with a history of breast cancer and osteoporosis visited our hospital with a chief concern of left lateral thigh pain for 2 weeks. She had been receiving PPI and monthly alendronate for 10 years for the management of osteoporosis. The patient had no obvious sensory disturbance or muscle weakness. She had no rest pain in the left lower extremity, but had pain during weight-bearing and walkingLCS is often difficult to diagnose as a large number of differentials can mimic this condition, including herpes zoster, peripheral arterial disease, peripheral neuropathy, , 9 and pAFF is a type of stress fracture called an insufficiency fracture that develops over time, most commonly after prolonged suppression of bone remodeling by the bisphosphonates . RepetitA systematic review found that the mean reported age range of patients with bisphosphonate-related AFF who had surgical treatment was 66.8–74.2 years . In the Although the presence of the dreaded black line on the initial radiographs is a radiologic feature indicative of stress fracture nonunion,  the dreaDelayed or missed diagnosis of AFF, especially in the case of an incomplete fracture, may negatively impact the patient’s prognosis, as seen in the two cases presented where the fractures both progressed from incomplete to complete. Banffy et al. reported that five of six incomplete AFFs progressed to complete AFF, and patients who underwent surgery for incomplete AFF had shorter hospital stays than those of patients with complete AFF . CarefulIn summary, we reported two cases of AFF mimicking lumbar radiculopathy. Clinicians should consider AFF as a differential diagnosis in older patients with LCS who are undergoing long-term bisphosphonate therapy and present with thigh pain.


Vitamin K-dependent (VKD) proteins undergo an unusual post-translational modification, which is the conversion of specific Glu residues to carboxylated Glu (Gla). Gla generation is required for the activation of VKD proteins, and occurs in the endoplasmic reticulum during their secretion to either the cell surface or from the cell. The gamma-glutamyl carboxylase produces Gla using reduced vitamin K, which becomes oxygenated to vitamin K epoxide. Reduced vitamin K is then regenerated by a vitamin K oxidoreductase (VKORC1), and this interconversion of oxygenated and reduced vitamin K is referred to as the vitamin K cycle. Many of the VKD proteins support hemostasis, which is suppressed during therapy with warfarin that inhibits VKORC1 activity. VKD proteins also impact a broad range of physiologies beyond hemostasis, which includes regulation of calcification, apoptosis, complement, growth control, signal transduction and angiogenesis. The review covers the roles of VKD proteins, how they become activated, and how disruption of carboxylation can lead to disease. VKD proteins contain clusters of Gla residues that form a calcium-binding module important for activity, and carboxylase processivity allows the generation of multiple Glas. The review discusses how impaired carboxylase processivity results in the pseudoxanthoma elasticum-like disease. Dietary vitamin K is used in virtually all tissues to activate vitamin K-dependent (VKD) proteins through the conversion of specific glutamyl (Glu) residues to carboxylated Glu (Gla) ,2. VKD pVitamin K was originally implicated as important to hemostasis by the hemorrhagic response of chicks fed a lipid free diet. Subsequent discoveries that were significant in understanding the role of vitamin K were its isolation and identification, the identification of VKD proteins, and the demonstration that these proteins undergo an unusual post-translational modification, which is the carboxylation of specific Glu residues to Glas ,2,3. The−/−mgp mice, who die shortly after birth due to massive arterial calcification and vascular rupture [−/−grp mice do not reveal a clear phenotype as in the case of the −/−mgp mice. The remaining nonhemostatic proteins, i.e., the TMG proteins, are distinct from all other known VKD proteins in having a single pass transmembrane sequence that localizes the Gla domain at the exterior of the plasma membrane. These proteins are expressed in a wide variety of tissues and have motifs suggesting roles in signal transduction [An even broader impact of vitamin K on human health has been revealed by the identification of nonhemostatic VKD proteins (Matrix Gla Protein (MGP), osteocalcin, Gla Rich Protein (GRP) and four transmembrane proteins (TMGs) C,D. MGP  rupture . Warfari rupture ,29. In h rupture . Mutatio rupture ,33,34,35sduction ,37; howeVKD proteins undergo the conversion of Glus to Glas in the endoplasmic reticulum during secretion  Figure . These pThe secretion of VKD proteins from the cell is impacted by quality control mechanisms in the endoplasmic reticulum, which ensure that only functional proteins and/or protein complexes are secreted A. RegulaProtein maturation in the case of the VKD proteins includes an additional, unique step, i.e., Gla production B. The spVKD protein carboxylation is a complex process that involves several enzymes. The carboxylase converts multiple Glus to Glas in the Gla domains of VKD proteins, which generates a calcium-binding module that is essential for activity ,45 Figu. The carVitamin K refers to a family of forms that comprise a single phylloquinone and multiple menaquinones. These forms all have the same 2-methyl-1,4,-naphthoquinone nucleus, which is attached to a polyisoprenoid side chain. The side chain is more unsaturated in menaquinones, which vary in the number of isoprenoid units. High levels of both phylloquinone and menaquinone decrease vascular calcification in rodent models . However2 to form Gla [The carboxylase is an integral membrane protein, which allows interaction with the hydrophobic vitamin K cofactor that is also membrane-embedded ,54. The form Gla  , which is used to prevent blood clotting in patients, for example who have mechanical heart valves, deep vein thrombosis or pulmonary embolism. Warfarin uncouples normal VKORC1 reduction, which disrupts full vitamin K epoxide reduction by inhibiting quinone reduction much more strongly than epoxide reduction C. How wa−/−nqo1 mice [The response of patients to warfarin is variable, and some show bleeding complications during warfarin therapy that are rescued by vitamin K. Large amounts of vitamin K quinone are administered, which obviates the need for VKORC1 recycling because the high levels of vitamin K quinone can support VKD protein carboxylation through a quinone reductase. There have been significant efforts to identify the antidotal quinone reductase that rescues bleeding. Warfarin resistant quinone reductase activity has been demonstrated in liver ,73,74, w−/− mice . Another−/− mice . However−/− mice , consist−/− mice . VKORC1 −/− mice ,81. Howe−/− mice ,83. FutuVKD proteins contain multiple Gla residues, which form a highly organized network of contacts with several calcium ions . The strThe presence of multiple Gla residues in VKD proteins raises the question of how the carboxylase accomplishes full carboxylation of the Gla domain. Other enzymes that act on substrates containing repetitive units have been shown to be processive , i.e., tCarboxylase processivity was studied using a novel challenge assay that mimicked in vivo conditions. A VKD protein-carboxylase complex  is incubated with a distinguishable VKD protein that is present in large excess over the complex A. In a pAnother important outcome of the studies came from rate analyses, which suggested that carboxylase processivity may not always result in the full carboxylation of VKD proteins. Full carboxylation of the Gla domain requires that the release of carboxylated VKD proteins is slower than the time to modify all Glas in the Gla domain, and we found that release is only 5–6 fold slower ,16. ThisThese studies raise new questions about VKD protein carboxylation, for example whether vitamin K nutriture or warfarin therapy impact processivity and consequent full carboxylation. Dietary vitamin K levels are low and vary significantly with the diet, and vitamin K levels control the rate of catalysis. Vitamin K deficiency may therefore impact processivity by altering the relative rates of catalysis versus release. In addition, vitamin K levels vary significantly in different tissues, raising the question of whether the efficiency of processivity is the same in all tissues. Warfarin therapy may also impact processivity, because warfarin inhibition of VKORC1 decreases the supply of reduced vitamin K and therefore lowers the rate of catalysis. Early studies on warfarin inhibition revealed heterogeneous forms of prothrombin ,91, and Impaired VKD protein carboxylation is associated with multiple pathologies that include bleeding, soft tissue calcification, chronic kidney disease, calciphylaxis, atherosclerosis, osteoarthritis and osteoporosis ,113,114.A large number of VKORC1 mutations cause warfarin resistance, which is the need for higher doses of warfarin to manage hemostasis . The reqCarboxylase mutations also cause disease, which includes VKCFD1 that is associated with severe bleeding. Like VKORC1, mutations that are essential for carboxylase activity  haveRegulatory regions of the carboxylase are largely unknown, and our studies revealed the importance of carboxylase processivity to human health. Combined heterozygosity of the carboxylase mutations Val255Met and Ser300Phe causes PXE-like, and mutant analysis using small peptides derived from VKD proteins did not explain the defect in the patient . The appThe studies raise new questions regarding the impact of impaired processivity on disease. One question is the functional consequence of defective processivity. The Val255Met mutant is responsible for the survival of the patient and yet generated partially carboxylated factor IX, indicating that full factor IX carboxylation is not absolutely required for function. Assessing the impact of impaired processivity on the activity of other VKD proteins should be informative. An interesting consequence of impaired processivity was the production of much higher levels of partially carboxylated factor IX and MGP by the Val255Met mutant than fully carboxylated VKD protein generated by wild type carboxylase . Higher VKD protein carboxylation is commonly assessed using antibodies developed against large Gla- or Glu-containing peptides derived from individual VKD proteins. This approach was originally developed using antibody against the hemostatic VKD protein prothrombin, which is referred to as PIVKA (for protein induced by vitamin K absence). The PIVKA assay has been valuable, for example as a biomarker for poor prothrombin carboxylation in hepatocellular carcinoma ,124. AntSome considerations are important for interpreting results obtained using the antibody approach. One consideration is that the antibodies do not necessarily inform on the extent of carboxylation. The antibody recognizes a Gla-containing sequence, however even a few Glas could elicit antibody reactivity. The antibodies are sometimes referred to as conformation specific, however the number of Gla residues required to induce the conformational change is unknown. This point is underscored by a recent study that analyzed the same Val255Met mutant that we showed is impaired in processivity. The study concluded that the Val255Met mutant has wild type levels of carboxylation, using a conformation specific antibody that recognizes Gla but did not reveal partial carboxylation . A seconMethods that have been developed to assess the extent of carboxylation and/or investigate individual Gla residues are Gla quantitation, Edman degradation and mass spectrometry. Gla quantitation involves hydrolyzing purified VKD proteins to amino acids, which are then separated and quantitated using HPLC . This apMass spectrometry is a powerful approach to study carboxylation because it can identify each Glu/Gla residue in the Gla domain of an individual VKD protein molecule. One approach for studying VKD proteins is liquid chromatography mass spectrometry, in which peptide digests are fractionated on a column, and then ionized and bombarded to sequence the peptide. Bombardment results in the loss of the Gla side chain before cleavage of the peptide backbone, which precludes obtaining sequence information; however, modification of the Gla residues by methylation prevents Gla cleavage and allows sequencing of Gla-containing peptides . This ap


The emergence of a new coronavirus presents a huge risk to public health worldwide and has spread widely amongst the human population. Since its emergence, the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is frequently evolving by mutation and genetic recombination to give rise to new viral variants. These emerging variants pose a challenge to existing COVID-19 management strategies and vaccine efficacy. Interruption of viral spread is required as the merging variants pose higher transmissibility than the previous ones. To achieve this, local protection of the respiratory tract with immunity is essential. Here, we advocate the use of pulmonary/inhalable vaccines to achieve this goal. Similarly, IgG is essential for protective immunity in the lower respiratory tract, but IgA is more critical in the nasal region [Since the emergence of “severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2)” in December 2019, immunization continues to be the foundation of healthcare today ,4. The Sl region ,23. Vaccl region ,25. In aTherefore, there seems to be an unrelenting need to create inhalation-based immunization strategies that follow the similar process of COVID-19; start of infection , advancement , and aggravation. Intranasal vaccines are known to prevent airborne diseases by providing multifront immunization effectively.+ CD8+ T cells and complementing serum neutralizing antibodies [While COVID-19 has encouraged the development of innovative vaccination formulations, a major fraction of the proteinaceous compositions presented practical difficulties with respect to temperature control. Investigation of inhaled vaccine administration should be carried out on the creation of stable dry vaccine dosage forms, which have the benefit of improved stability and may thus be kept for longer durations without decomposition at less stringent temperatures. Aerosolized vaccinations should be carefully explored as a means of combating highly contagious viral infections such as COVID-19 . The Ad5tibodies . A phaseWe believe that the spectrum of vaccination development should be expanded to include aerosolized dry powder compositions due to the many benefits of dry vaccines . Its dryness may significantly improve the stability and longevity of vaccines. Nano/microparticulate forms of vaccines may be breathed using portable inhalers or nasal spray, eliminating the requirement for skilled medical staff and simplifying mass immunization programs . Researc


Ostrom et al., 2015). Furthermore, the vast majority of these tumors recur. With limitations in the ability to monitor using clinical and radiographic methods alone, minimally invasive methods such as liquid biopsy will be crucial to our understanding and treatment. Liquid biopsy of blood, urine, and cerebrospinal fluid (CSF) can be used to sample cfDNA, ctDNA, RNA, extracellular vesicles, and tumor-associated proteins. In the past year, four seminal papers have made significant advances in the use of liquid biopsy in pediatric brain tumor patients (Malignant primary brain tumors are the most common cancer in children aged 0–14 years, and are the most common cause of death among pediatric cancer patients. Compared to other cancers, pediatric brain tumors have been difficult to diagnose and study given the high risk of intracranial biopsy penetrating through vital midline structures, where the majority of pediatric brain tumors originate (patients . In this Ostrom et al., 2020). Unfortunately, diagnosis via clinical evaluation and MRI imaging is uncertain, with a differential diagnosis including abscesses and tumor variants requiring discordant treatments. Therefore, current clinical decision-making requires surgical biopsy or resection for histopathologic diagnosis. Even the most minimally invasive option–stereotactic needle biopsy–puts the patient at risk of side effects of general anesthesia, intracranial hemorrhage, neurologic deficit, and death . It has previously been shown that neuropathologic diagnosis via stereotactic biopsy specimen versus tumor resection specimen of the same lesion differed in approximately 40% of cases . The development of a safer, more accurate diagnostic tool would revolutionize the treatment of CNS tumors.The most common cause of death among pediatric cancer patients aged 0–14 years are malignant primary brain tumors . A reliable, non-invasive method for differentiation between progression and pseudoprogression would obviate the need for repeat biopsy and facilitate early treatment.Similarly, the current paradigm of clinical and radiographic tumor monitoring during and following treatment is ineffective in guiding clinical decision-making due to radiographic changes with poor sensitivity and specificity for true tumor growth. So-called pseudoprogression–radiographic tumor growth and/or clinical deteriorationis a result of treatment-effect: Inflammatory changes which can be controlled with steroids .Histopathologic criteria once dominated brain tumor diagnosis, however, the newer WHO guidelines are progressively shifting toward molecular and genomic parameters . A comprGiven its ability to transcend the safety concerns and diagnostic limitations of stereotactic biopsy, the development and application of liquid biopsy in clinical CNS tumor management is critical. In this review, we will discuss the latest technologies in biofluid isolation and interpretation, as well as previously identified biomarkers for detection and tracking of various pediatric CNS tumors. Finally, we will discuss the applicability of these techniques and future integration into the current clinical system.Due to a lower total biofluid volume in pediatric patients, the ability to isolate and identify genetic and molecular components from small samples is crucial. Here we will discuss general principles of liquid biopsy in pediatric brain tumors and showcase the feasibility of tumor detection using biofluids .PCR (polymerase chain reaction) amplifies DNA using a combination of primers, dNTP, and DNA polymerase. Specifically, quantitative PCR captures real-time amplification of targets using fluorescently assigned probes. ddPCR furthermore allows for unbiased amplification of a known target by isolating reactions in each of thousands of droplets . ddPCR ivia the addition of targeted primers and probes. NGS circumvents this issue with its capacity to seek innumerable unknown genetic alterations . A recent study utilized the NGS Memorial Sloan Kettering-Integrated Molecular Profiling of Actionable Cancer Targets (MSK-IMPACT) assay. Using CSF from pediatric patients with varied histologies and a sequencing depth of 2x to 1,368x, researchers screened patients with existing somatic alterations  sequences by synthesis, in which a 500-base-pair adaptor is ligated and fluorescent dNTPs are added to the strand to determine the sequence of the target strand . Illuminerations .Nanopore sequencing utilizes miniature pores to sequence strands of DNA . As a stvia liquid chromatography, which separates compounds in a sample based on interactions between mobile and stationary phases. Mass spectrometry is then utilized to convert molecules to the ionized state allowing compound identification by measuring the speed of movement through a vacuum chamber . With only 10 μL of plasma, 862 proteins were identified within 12 h using LC-MS . Using CSF LC-MS, six proteins were successfully identified that could discriminate the metastatic status of CNS tumors in children compared to controls .Proteomic characterization of CNS tumors is possible Guntner et al., 2020).LC-MS can also be used to analyze the metabolites in CSF created by the intake of anti-cancer drugs to assess penetrance beyond the blood-brain barrier (BBB), important for identifying new therapies for clinical trials. A study of seven oral anti-cancer drugs in pediatric CNS tumor patients successfully demonstrated the ability of LC-MS to identify medications with the highest BBB penetrance . Circulating tumor DNA (ctDNA) are mutation-carrying fragments released by tumor cells. Both total cfDNA quantification and the mutations identified in ctDNA can be used as biomarkers for tumor diagnosis and progression. Due to its short half-life of 16 min to 2.5 h cfDNA could be a real-time marker of treatment effect and measurable residual disease (MRD) .Cell-free DNA (cfDNA) are 50–200 base-pair DNA fragments that are released from cells Pan et al., 2019). Our group demonstrated correlation between the concentration of characteristic mutant K27M copies in CSF ctDNA and contrast-enhancing tumor area on MRI, as well as with cell proliferation in an in vitro model . We further applied this technique in a larger cohort as part of a prospective clinical trial, demonstrating that change in H3.3K27M variant allele fraction (VAF) in both CSF and plasma was associated with increased progression-free survival. VAF increase of >25% was found to precede tumor progression in many cases, and cases of radiographic progression without an increase in VAF were later found to reflect pseudo-progression. These data show that CSF or plasma ctDNA H3.3K27M VAF could be a reliable biomarker for prediction of progression and for differentiation from pseudoprogression , low-coverage WGS detected MRD in 54% and 85% of patients with localized and metastatic disease, respectively . Among 6microRNAs (miRNAs) are non-coding molecules 18–24 nucleotides in length . They arEGFR(v)III amplification in pediatric high-grade gliomas  miRNA expression changes have been identified that directly regulate tumorigenic MB pathways including SHH and WNT . These bExtracellular vesicles (EV) are small, membrane-bound structures secreted from both normal and cancerous cells. EVs have been found to hold tumor-specific DNA, various RNAs, and proteins . Becausein vitro. Isolation of EVs from the cell media supernatant of 3 DIPG and 4 MB cell lines identified EV-derived miRNAs unique to DIPG and MB respectively . This has further been shown to be reproducible using liquid biopsy, with the ability to identify targetable mutations, monitor therapeutic response, and follow tumor evolution . Spatial subclones with unique genotypes and phenotypes have been identified in pediatric GBM and DIPG . In an adult diffuse glioma pilot study, 85% of tumors were successfully diagnosed via CSF ctDNA sequencing with a 7–10 days turnaround . Liquid biopsy miRNA markers for medulloblastoma have been identified for differentiation from controls with high specificity . A correlate in pediatric brain cancers could play an important role in post-treatment monitoring and early treatment of recurrence. Serial liquid biopsy in addition to serial MRI may reveal new genetic targets for precision chemotherapy .Our group demonstrated that change in CSF cfDNA H3K27M variant allele fraction predicted treatment response and tumor progression following ONC201 treatment in diffuse midline glioma . The samvia lumbar puncture in children is complicated. Furthermore, it has been shown that CSF sampling more proximal to an intracranial tumor may have increased sensitivity for CSF ctDNA detection . Studies have also found that placement of a fourth ventricular catheter for even greater proximity to a posterior fossa tumor is safe .Brain biopsy, the gold standard of pediatric brain tumor diagnosis, is a risky procedure with unreliable diagnostic value. Liquid biopsy could be a minimally invasive, definitive method to diagnose, monitor, and treat these lethal cancers. CSF procurement Vitanza et al., 2021).LP is the safest, least invasive method to obtain CSF during initial brain lesion workup. However, in patients who undergo surgical resection, an alternative CSF procurement method could involve placement of an Ommaya reservoir and peritumoral or intraventricular catheter. Lateral and third ventricular catheters are routinely placed by neurosurgeons for CSF diversion or intrathecal chemotherapy. Still, reservoir placement for tumor monitoring and CAR-T therapy in pediatric patients has only recently become standard of care . Other rare complications include hemorrhage during placement, clogging, and superficial local CSF effusion. Given safety of placement, the potential benefits for longitudinal disease monitoring and treatment guidance far outweigh the benefits.Given that most pediatric tumors present in the posterior fossa, hydrocephalus is a common issue. More than 60% of children with DIPG require surgical intervention for relief, many of whom require shunt placement. In a review of several studies of shunt catheters in DIPG, the overall infection rate was <1% . In chilBlood sampling is innocuous when compared with CSF extraction. The sensitivities of sequencing technologies used to study biofluids, specifically low-coverage WGS and targeted Nanopore NGS, are increasingly improving. As the ability to detect nucleic acid and proteomic biomarkers from liquid biopsies improve, the emphasis will transition from CSF to serum studies. We have already begun to demonstrate that plasma can be used to direct clinical decision-making without awaiting identifiable radiographic progression .Further research is required in the characterization and standardization of biomarkers found in CSF and other biofluids, which could represent the next paradigm shift in clinical management of pediatric brain tumor patients.


The dynamic interplay between virus and host plays out across many interacting surfaces as virus and host evolve continually in response to one another. In particular, epitope-paratope interactions (EPIs) between viral antigen and host antibodies drive much of this evolutionary race. In this review, we describe a series of recent studies examining aspects of epitope complexity that go beyond two interacting protein surfaces as EPIs are typically understood. To structure our discussion, we present a framework for understanding epitope complexity as a spectrum along a series of axes, focusing primarily on 1) epitope biochemical complexity  and 2) antigen conformational/dynamic complexity . We highlight additional epitope complexity factors including epitope tertiary/quaternary structure, which contribute to epistatic relationships between epitope residues within- or adjacent-to a given epitope, as well as epitope overlap resulting from polyclonal antibody responses, which is relevant when assessing antigenic pressure against a given epitope. Finally, we discuss how these different forms of epitope complexity can limit EPI analyses and therapeutic antibody development, as well as recent efforts to overcome these limitations. EPIs are key aspects of the dynamic interplay between the virus and the host immune response to neutralize the virus.Viral surface proteins are typically the immunodominant antigens that are targeted for antibody-mediated neutralization by the humoral immune response by the host. These viral proteins present numerous surfaces known as epitopes which are recognized by antibodies that are generated by the host immune system to specifically bind to these virus epitopes Host antibody responses upon viral infection vary widely depending on the virus and the host’s exposure history to the virus, homologous viruses, and vaccines. Hosts that have been previously infected or vaccinated typically possess neutralizing antibodies (nAbs) against vulnerable virus epitopes which protect the host from infection upon viral exposure, with the nAb titer often correlating with the degree of protection against future infections , 2. Howevia Fc-mediated mechanisms, poorly-neutralizing antibodies are undesirable as they may not protect the host from future exposures and thus lead to reinfection, though non-neutralizing antibodies can still play key roles in protection via Fc function  that retain functionality across many variants of the same virus as well as cross-neutralize evolutionarily-related viruses  or matured (resulting from accumulation of somatic mutations within memory B cells) antibodies to neutralize viruses bearing mutated or homologous epitopes –20, wher viruses –30. Ther viruses –36. LikeA variety of experimental approaches and computational tools have been developed to map virus antigenic landscapes, which have become increasingly valuable during the global response to address SARS-CoV-2 and subsequent waves of variants of concern. Experimental approaches include deep mutational scanning of immunodominant domains , 37–40, Epitopes on pathogenic surface proteins have varying levels of conformational, dynamic, and post-translational complexity, all of which can have significant effects on the binding, specificity and neutralization potential of an antibody targeting that epitope. Conformationally, epitopes range from relatively simple, in that they are comprised of a linear stretch of amino acids on a monomeric protein domain, to complex, in that they are discontinuous and sample distinct conformations in a monomeric or even multimeric (quaternary) assembly of protein domains on the viral surface. Additionally, epitopes on viral surface proteins are not static in time. Many undergo significant conformational changes, including rearrangement of entire protein domains across the various stages of the viral life cycle. Finally, post-translational modifications such as glycosylation of viral surface proteins can either mask the underlying protein epitope surface or be part of epitope surfaces that are targeted by host antibodies as epitope constituents.direct dependence on the underlying protein surface (Glycans as epitope constituents have been most widely appreciated and interrogated through the study of HIV glycoprotein 120 (gp120), wherein researchers have sought to specifically induce and characterize the development of anti-glycan Abs , 62. Suc surface –67. A va surface , 68–70,  surface –73, and  surface –76. 2G12 surface  that simBeyond inclusion of glycans, epitope complexity can result from epitope secondary, tertiary, and quaternary structure which can present an epitope in multiple formats depending on viral capsid arrangement , 78–80. In this review, we describe distinct epitope features driving epitope complexity, including biochemical complexity resulting from factors such as glycosylation, conformational complexity resulting from antigen quaternary structural dynamics, and epistatic and allosteric complexity resulting from epitope secondary and tertiary structure. We include specific examples of each form of epitope complexity, and describe recent publications on efforts to model, analyze, and engineer epitope-paratope interactions that accommodate epitope complexity. Specifically, we present examples of network and structure-based analyses of complex viral epitope surfaces from the standpoint of improving design and repurposing of therapeutic antibodies and mapping antigenic drift, as performed by us and others. These approaches include functional characterization of a cross-neutralizing Henipavirus antibody , developThe range of epitope complexity presented on viral surface proteins drives ease of characterizing epitope-paratope interaction (EPI), availability of standard methods and tools to analyze the EPI and engineer antibodies against the epitope, and the amount of existing biological information/context required for such endeavors. To frame our perspective on epitope complexity, we first illustrate epitope complexity along two axes: 1) biochemical complexity involving factors such as glycosylation, and 2) conformational complexity involving epitope secondary, tertiary, and quaternary structure as well as epitope dynamics  is a zoonotic RNA virus of the Paramyxoviradae family , which ivia ABodyBuilder  despite site 406 residing outside of the epitopes of all three Abs. While E406W is likely to be one of the more significant allosteric escape mutations on SARS-CoV-2 RBD, similar mechanisms may play a more local role for many escape mutations, especially when occurring in combination with other mutations as observed for the Omicron BA.1 and BA.2 subvariants. For an example of epistatic escape effects—or the synergistic effect resulting from combinations of mutations—consider the escape of BA.1 from antibody ADG20 (Adintrevimab). It was predicted that broad-spectrum and potent anti-SARS-CoV-2 antibody ADG20 would retain activity against the BA.1 Omicron sub-variant based on an analysis that ADG20 targets a highly sequence-conserved epitope surface and that the individual Omicron BA.1 mutations are not individually associated with escape in vitro  network analysis , which qvia a novel interaction between L/F371 and the N343 N-glycan  trajectory. To the best of our knowledge such network+MD analyses have yet to be performed, though are likely to be a fruitful research avenue. A contemporary example of epitope dynamics driving such complexity wherein current tools are limited in their application are the SARS-CoV-2 Omicron sub-variant mutations S371L and S371F mutations. S371L/F were recently observed to result in substantial escape from all four classes of anti-RBD antibodies in pseudoviral experiments , 49. YetN-glycan . More sp3 glycan , in whic3 glycan , which hStill, AAI and other escape-mapping techniques have proved valuable for rapidly assessing emerging SARS-CoV-2 variants and their escape mutations, in large part due to their modeling of indirect effects mediated by higher-order structure. We recently applied AAI to rapidly predict the impact of the Omicron BA.1 mutations on the set of clinically-authorized therapeutic antibodies in the days immediately following the first observation of BA.1 . We founindirect networking term of AAI substantially increases the modeling utility of the technique. Indeed, it is clear that models considering only direct interactions between the epitope and paratope are unable to identify all antibodies escaped by a given variant. Further, combining the direct and indirect features into a single Total Networking metric, which AAI uses for its primary prediction, produces a logistic relationship between modeled epitope perturbations and experimentally measured escape that appears to have predictive value. This comparison of direct, indirect, and total networking metrics broadly suggests that indirect effects—which result from various features of epitope complexity—are critical for modeling SARS-CoV-2 variant escape from RBD-directed antibodies.Toward a more rigorous investigation of AAI for modeling the escape effect of SARS-CoV-2 VOC on therapeutic antibodies, we present next a comparison of 28 previously-published antibody-VOC escape interactions . This coIn the months since the computational and experimental data in via confirmation of biologically-correct glycosylation  on recombinant spike or RBD antigens. Second, in the AAI survey of variant-antibody interactions, we identified the BRII-196 (Amubarvimab) and Beta variant interaction to be incompletely modeled. Examining the direct and indirect features for this interaction, we found that the predicted escape results from a high direct networking perturbation. This is consistent with structural analysis of the interaction, which shows a hydrogen bond and hydrophobic interactions between paratope residues Y33, Y52 and epitope residue K417, suggesting that the K417N mutation on the Beta variant is likely to directly perturb the interaction. Yet, a structure between BRII-196 and the Beta variant has been solved, and indicates that the mutated N417 residue forms a new hydrogen bond with paratope residue Y52  targets the N-glycan at residue N343 on RBD, yet as described earlier in this review epitopes including N-glycans are complex and poorly modeled by existing methods. Computational and experimental approaches for investigating S309 against variants are therefore limited unless they are validated in their ability to faithfully capture interactions with the S309 proteoglycan epitope, for example, idue Y52 . This ouIn summary, epitope analysis that considers network complexity resulting from higher-order structures enables better modeling of allosteric and epistatic effects and offers improvement over conventional EPI annotations that consider only direct epitope-paratope interactions. These features can be captured by approaches such as the AAI indirect networking metric. However, such approaches that consider these aspects of epitope complexity can still be limited as a result of other aspects of epitope complexity which they may not capture such as glycan-protein interactions (as in the case of S309) and antigen structural dynamics .via modulation of fitness landscapes . This definition of epitope surface at the level of protein sequence at interface of antibody-antigen interaction has shaped understanding of epitope similarity between antigens and antibody engineering for targeting or enhancing affinity to a given antigen.However, as highlighted in this review epitopes on viral surface antigens have several layers of complexity in epitope-paratope interactions owing to 1) the quaternary assembly and higher-order structure of protein domains on the viral surface, 2) protein glycosylation including at clustered sites (that lead to predominance of non-self surface glycans such as high-mannose type structures), and 3) large conformational transitions of the surface proteins owing to their role in both receptor binding and membrane fusion during various stages of maturation in the viral infection cycle.In this review, we discuss aspects of epitope complexity that must be considered when targeting certain desirable viral antigens and when modelling and predicting viral evolution. As an example, the cross-neutralizing antibody engineering for Nipah virus case study describes a path for rapid response in the event a Henipavirus (or other virus with well-characterized homologous EPIs) were to emerge as a pandemic threat. This path would involve 1) rapid experimental testing of all antibodies known to target related G proteins to identify template cross-neutralizers, 2) structure prediction of the novel G protein and the top binding template antibodies, 3) rational computational design to enhance affinity and specificity of the templates against the novel NiV-like virus.Our review also points to the role of protein glycosylation in going beyond masking sequence epitopes to generating novel glycoepitopes that have been underappreciated in the context of defining epitope targets for neutralization by antibodies. This is evident from our approach to define a glycoepitope model for the 2G12 antibody that specifically recognizing an N-glycan cluster motif (without any contact with the amino acids on the protein) that can be applied to search other pathogens for similar motifs thereby providing potential repurposing targets for 2G12. A similar rapid repurposing effort might prove fruitful in the case of an emerging influenza A H3N2 lineage bearing a new N-glycan proximal to the HA receptor binding site.Understanding epitope complexity, particularly in terms of epitope networks and polyclonal epitope space, is also imperative for selecting and engineering therapeutic antibodies. As observed across SARS-CoV-2 VOCs, antigenic pressure resulting in escape mutations appears to reflect overlap of immunodominant antibody epitopes, regardless of whether a given VOC has emerged as it navigated population-level immunity or within a chronic infection. This has resulted in two formats of escape VOC emerging in the pandemic thus far: 1) Beta/Gamma-like VOC with 2-3 mutations in orthogonal and immunodominant epitope regions such that they efficiently escape from a significant fraction of polyclonal antibodies, and 2) Omicron-like VOC which feature a greater number of mutations that result in both escape breadth  and depth (mutations accumulated within epitopes).In both VOC cases, many therapeutic antibodies isolated from convalescent individuals that heavily weight conventional design objectives such as very high neutralization potency were easily escaped. Instead, antibodies such as S309 with lower potency, but targeting an epitope distinct from population-immunodominance profiles are observed to have preserved function. These findings and our modeling therefore suggest that therapeutic antibodies should be designed as cocktails targeting orthogonal epitopes with lesser public immunodominance toward long-lived clinical relevance. Epitopes under high antigenic pressure in the population resulting from natural infection and vaccination should be avoided, even if these antibodies target an epitope that is less common and highly-conserved such as the epitope targeted by ADG20 that was conserved across many CoVs, as adjacent/overlapping epitopes may still exert sufficient pressure to prompt mutations with a large cumulative indirect effect on the conserved epitope. This further highlights the value of rational antibody engineering, as products can be by design distinct from common natural antibodies.In the context of the epitope complexity highlighted in this review, viewing epitope surface just as an amino acid footprint at the sequence level has led to many challenges that point to key gaps in this view. Such sequence-based analyses also tend to simplify epitope similarity on basis of sequence conservation ignoring nuanced impact of structural constraints on mutability imposed by higher-order structure, glycosylation, and conformational dynamics. For example , 142, thAll authors conceived the work. NM and TC performed the analyses. NM and RR wrote the manuscript. All authors contributed to the article and approved the submitted version.NM was supported in part by T32 ES007020/ES/NIEHS NIH.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Glechoma longituba, were grown adjacent to different genetically related neighbors (clone kin / close kin / distant kin) in different nutrient conditions (high / medium / low), or with only root exudates from pre-treatment in culture solution. By comparing competitive traits, we found that: (1) kin recognition in G. longituba was enhanced with closer genetic distance; (2) the outcomes of kin recognition were influenced by the extent of nutrient shortage; (3) kin recognition helped to alleviate the nutrient shortage effect; (4) kin recognition via root exudates affected only below-ground growth. Our results provide new insights on the potential for manipulating the outcome of kin recognition by altering neighbor genetic distance, nutrient conditions and recognition ways. Moreover, kin recognition can help plants mitigate the effects of nutrient shortage, with potential implications in agricultural research.Kin recognition might help plants decrease competitive cost and improve inclusive fitness with close genes; thus it might interact with environmental factors to affect communities. Whether and how various factors, such as the genetic distance of neighbors, environmental stressors, or the way a plant recognizes its neighbors, might modify plant growth strategies remains unclear. To answer these questions, we conducted experiments in which ramets of a clonal plant,  Kin selection benefits related genes and improves inclusive fitness; this altruism is favored in two ways: kin recognition and viscous populations . Most plPrevious studies have examined plant kin recognition among different related neighbors , but theSorghum vulgare found that kin-benefit interactions in nutrient-poor soils were less pronounced than in nutrient-rich soil  and to receive/emit signals from the environment, and root is the major organ of below-ground performance . Root exthaliana  and Dist spicata . But aft spicata  and phot spicata , were alGlechoma longituba, and investigate how factors like neighbor genetic distance, nutrient stress, or ways of recognition affected plant growth. In our experiment, the plants had no initial below-ground parts, and the effects we detected as changes in plant architecture were most pronounced in the below-ground parts. Accordingly, we focused mainly on morphological traits below-ground and the relative growth rate (RGR) index of above-ground plant parts. Specifically, we tested four hypotheses: (1) kin recognition in G. longituba is stronger with closer genetic distance; (2) the outcome of kin recognition is influenced by nutrient shortage; (3) kin recognition helps to alleviate the effect of nutrient shortage; (4) kin recognition via root exudates can affect the growth of the whole plant.In the current study, we conducted a greenhouse experiment to explore kin recognition in a clonal plant, G. longituba is a normal stoloniferous clonal plant species in China. Each G. longituba ramet has two opposite leaves and produces roots when stolons touch the ground.G. longituba used in our joint experiments were collected from Fenghuangzuigou  and Hamagou  in Qinling Mountains, which are 1.3 km apart with a valley between them. There were two different plots (1 × 1 m2) in Fenghuangzuigou which were more than 10 m apart and considered plot A1 and A2, while only one plot (1 × 1 m2) in Hamagou was considered plot B. We calculated the genetic distance of several genet samples collected from plot A1/A2 and plot B  of kin neighbor for genetic distance treatment. All genets were collected on 19th March 2016 and then planted in a greenhouse for 6 months before formal experiments. Ramets chosen in all experiments were seedlings that had not touched culture media so did not have below-ground growth at the beginning of our experiments.Genets of d plot B . The anach other . So rameG. longituba discriminates different related kins and how factors like genetic distance, nutrient stress, or the way plant recognizes its neighbor modified outcome of kin recognition  do not have significant difference in their growth performance , close (target plant was from genets in plot A1 and its neighbor was from genets in plot A2), and distant (target plant was from genets in plot A1 and its neighbor was from genets in plot B) under a range of decreased nutrient levels (from high to medium to low). Each level had 7 replicates. Paired ramets were in similar size and the planting pots were full of high/medium/low nutrient solution (400 ml 100%/25%/6.25% Hoagland). The two ramets were placed at two diagonally corners of the pot to keep them separate during the experiment and avoid space competition. High/medium/low nutrient solution was added to each pot at the beginning and every 2 days during the experiment to maintain it at 400 ml  with the corresponding culture solution (400 ml) for 10 days. During this time, the high nutrient solution (medium/low nutrient solution in the other two nutrient level treatments only) would be added in each pot every 2 days to constantly maintain the volume of growth medium at 400 ml.The At the beginning and end of our experiment, fresh biomass, leaf area, and petiole length of each ramet were measured. And after experiments, length of the longest primary root, number of lateral roots were also measured. Then, specimens were separated into root, petiole, and leaf. These organics were dried at 60°C in an oven for 72 h and weighed separately.Considering there was no initial below-ground of all ramets, we used one-way ANOVA to do multiple comparisons among groups and analyze the effects of neighbor relatedness on below-ground performance in all treatments, including root biomass, length of the longest root, and number of lateral roots .The ramets in our experiment had initial above-ground part , so to dRGR = (Wt-Wi)/Wipost hoc test for these variables.where Wt is the final leaf area/petiole length, Wi is the initial/leaf area/petiole length. Then we used one-way ANOVA to do the Then we used generalized linear mixed-effects modeling (GLMM) to test main effects of the two factors we focused on in our experiment (nutrient level and neighbor kinship), and their interactive effect on ramet growth performance we mentioned above.All data were analyzed with SPSS 25.0 software. The data used in figures were all original data.Not only neighbor kinship has significant effects on ramet biomass traits and morphological traits , but nutG. longituba showed significant differences in most competitive traits between growing next to clone kin and growing with close or distant kin, while the difference between growing with close kin or distant kin was not statistically significant (Under the high nutrient treatment, nificant . MoreoveG. longituba showed more obvious discrimination among kinship levels: there were significant differences among growing with clone kin vs. close kin vs. distant kin in all growth traits except root length, and the outcome of kin recognition was stronger than under the high nutrient treatment (Under the medium nutrient treatment, reatment .G. longituba showed little difference among the three types of related kins, and no clear kin discrimination was detected (Under the low nutrient treatment, detected .G. longituba grown with clone kin showed the least change in most traits measured when the nutrient level was decreased, while ramets grown with close kin changed more and those grown with distant kin changed the most (Nonetheless, the most .G. longituba might have multiple ways to recognize neighbor identity, and the responses depended on the ways of recognition.Below-ground architectural traits of ramets in the root exudates treatment showed a similar trend to ramets grown under high/medium nutrient conditions in the genetic distance × nutrient level treatments . Both thOur results confirmed the first three of our hypotheses, in that the experiments showed that the outcomes of kin recognition were modulated by the genetic distance of neighbors and the nutrient conditions. Specifically, although root exudates were able to mediate kin recognition, the responses depended on multiple factors, and root exudates mediated only below-ground outcomes of kin recognition.G. longituba showed kin recognition with detectable outcomes both above and below ground under high nutrient conditions (G. longituba invested less in competitive morphological traits both above and below ground when the genetic distance between neighbors was closer. The lower RGR of leaf area and petiole length indicated less-successful competition for light (G. longituba competed less when the genetic distance of kin neighbors was closer, the discrimination between growing next to close kin and distant kin was not statistically significant. This might be because both the target plant and its neighbor were growing under favorable conditions, and the response to kin recognition might exert little effect on promoting population fitness; thus, while the plants might recognize different kins, this led to less-pronounced outcomes.Similar to most previous studies, nditions . Moreoveor light , while for light . Overallor light . We alsoG. longituba performance when growing with close kin or distant kin under high nutrient conditions; when the nutrient condition was decreased to medium, G. longituba statistically distinguished all clone/close/distant neighboring kins, revealing a stronger expression of kin recognition under the lower nutrient conditions. However, when nutrient levels were decreased to low, the outcome of kin recognition seemed to be masked by nutrient competition. This might explain why two recent studies on kin recognition and soil fertility found different results for plant performance with regard to kinship of neighbors under lower fertility (When the nutrient conditions were reduced from high to medium and even low levels, we found the outcome of kin recognition did not change in a pattern that correlated with nutrient levels. As mentioned above, there was no significant difference between ertility . The difertility . Taking G. longituba to kin recognition fluctuated under different nutrient conditions, we wondered whether growing next to closer kins might help plants improve their tolerance for low nutrient conditions. Many studies have shown that plants can synthesize information from their neighbors and nutrients and then respond to this information (G. longituba was affected less by adjacent clone/close kins when grown under low nutrient conditions, indicating that growing with close genetic neighbors might buffer the effects of severe nutrient deficit. This result is similar to a previous study that examined kin selection in inter- and intra-specific competition, and suggested that kinship might influence plant growth (Because the response of ormation . A recenormation , suggestt growth . Generalt growth . Our stuG. longituba can recognize different genetic relatedness by ways of root exudates. But the effect was not exerted on the whole plant in our experiment, suggesting that kin recognition responses might be influenced by the method of plant recognition. In the genetic distance experiment, a target plant would not only have physical contact with its neighbor, but might also obtain information from other above-ground signals like volatile chemical cues (G. longituba was able to recognize different kins by root exudates alone, but if there were no other kinship signals, the response was exerted weakly only below-ground and not throughout the plant. The different response in above-ground growth between the genetic distance treatment and the root exudates treatment indicates that above-ground and below-ground signals modulated the kin recognition response together, and the overall outcome was the result of integrating multiple ways of recognition. In summary, G. longituba can recognize kinship by different mechanisms, and the responses are affected by those mechanisms; thus kin recognition is a complex and sensitive process (Previous studies have shown that root exudates can mediate identity recognition , and ourcal cues  and photcal cues . By cont process . The dev process  should eG. longituba revealed that individual plants can recognize intra-specific kinship levels more accurately than we thought before, and the outcome of that recognition is strongly influenced by nutrient conditions. Moreover, growing alongside close genetic neighbors might help plants mitigate the effects of nutrient shortages. There are diverse mechanisms by which plants recognize their kin; these mechanisms might also determine the response to kin recognition. Our findings suggest the outcomes of kin recognition could be manipulated in various ways, such as adjusting environmental conditions, selecting the kinship in populations, or controlling the ways plants recognize and respond to their neighbors. By studying kin recognition abilities in specific plant species in crops, ornamental plants, medicinal herbs, etc., we may make plants improve their investment in seeds, fruits, flowers, leaves, or other organs we need by taking control of different factors mentioned above. And benefits by growing with close kins could even improve plant tolerance under unfavorable conditions. Therefore, further studies on wider ranges of kinships, species, and other factors would help us better understand kin recognition in different plants, and new techniques would enable us to explore more details of kin recognition mechanisms, these relevant studies would provide new potential applications on agriculture, forestry, and environmental protection.In conclusion, our investigation of kin recognition in The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.YF performed the experiment and wrote the article. YZ analyzed the data. RZ revised the draft and finalized the manuscript for submission with YF and MY. All authors contributed to the article and approved the submitted version.


Background: Antibiotic resistance is pervasive in the Veterans’ Affairs (VA) healthcare system, with rates of fluoroquinolone and trimethoprim–sulfamethoxazole (TMP/SMX) resistance approaching 30% in E. coli urinary isolates. The efficacy of antimicrobial treatment is critically dependent on the susceptibility of the infecting pathogen; however, prescription decisions are often made empirically in practice. We analyzed susceptibility profiles of enteric gram-negative rods  from clinical urine cultures collected from ambulatory patients receiving care in VA clinics and emergency departments. Our goals were (1) to develop a predictive model to support choice of empiric antibiotics pending results of susceptibility testing and (2) to examine the relationship between past antibiotic exposures and susceptibility profiles to enhance understanding of antibiotic selective pressure. Methods: We obtained 265,076 positive cultures from 157,422 unique patients from 2015 to 2020. We trained random forest multinomial classifiers to estimate the risk of a positive urine culture isolate being resistant to the multinomial outcome: fluoroquinolone, TMP–SMX, cephalosporin, or any combination of these 3 agents. Data sources evaluated for model generation included demographics, comorbidities, trend and seasonal terms, treatment history for multiple antimicrobial treatments summarized using number of prescriptions in weekly intervals, and sample history summarized by number of resistant and susceptible cultures in weekly intervals. Using 5-fold cross validation, we assess the performance of the clinical prediction using the area under the receiver operating characteristic curve (AUC) for each multinomial outcome. In addition to prediction, we modeled the direct effect of treatment on resistance using multinomial group lasso (MGL). This method allows variable selection in variable groupings, such as all variables related to the fluoroquinolone treatment history, which allowed us to assess the effect of a patient’s complete course of treatment on resistance. Results: In cross-validation analysis, our random forest model was best at predicting outcomes with fluoroquinolone resistant phenotypes compared to non–fluoroquinolone-resistant phenotypes  treatment is a stronger indicator of a future resistant phenotype than cephalosporin or penicillin.als were  to develals were  to develFunding: NoneDisclosures: None