Cardiac cell-based therapy represents a promising approach for cardiac repair. However, one of the main challenges is cardiac arrhythmias associated with stem cell transplantation. The current review summarizes the recent progress in model systems for addressing mechanisms of arrhythmogenesis in cardiac repair.Animal models have been extensively developed for mechanistic studies of cardiac arrhythmogenesis. Advances in human induced pluripotent stem cells (hiPSCs), patient-specific disease models, tissue engineering, and gene editing have greatly enhanced our ability to probe the mechanistic bases of cardiac arrhythmias. Additionally, recent development in multiscale computational studies and machine learning provides yet another powerful tool to quantitatively decipher the mechanisms of cardiac arrhythmias.Advancing efforts towards the integrations of experimental and computational studies are critical to gain insights into novel mitigation strategies for cardiac arrhythmias in cell-based therapy. Cardiovascular disease is the leading cause of morbidity and mortality worldwide and causes more deaths than all cancers combined . DespiteAnimal models have been extensively developed for mechanistic studies of cardiac arrhythmogenesis •, 11–13,The current review will summarize the recent progress in model systems for addressing mechanisms of cardiac arrhythmogenesis, and serve as a discussion platform to gain insights into mechanistic underpinnings and novel mitigation strategies for arrhythmogenesis in cardiac repair.Numerous species have been utilized to study the underlying mechanisms of arrhythmogenesis, ranging from small animals such as zebrafish to large animal models such as pigs and nonhuman primates Fig. . AlthougElectrical and structural remodeling in pathological cardiac hypertrophy has been shown to increase patient’s susceptibility to ventricular arrhythmias (VAs) and sudden cardiac death (SCD). Electrical remodeling in cardiac hypertrophy is well documented and includes action potential (AP) prolongation  and condCoronary artery disease (CAD), leading to myocardial ischemia and infarction, is a frequent cause of cardiac arrhythmia. Cardiac ischemia and infarction result in complex electrophysiological remodeling •. In murAtrial fibrillation (AF) is the most prevalent cardiac arrhythmia seen clinically . A commoHeart failure (HF) occurs when the cardiac output is no longer able to meet the metabolic demands of the body, and is frequently associated with cardiac arrhythmias . Animal TGFB1 in goats, and mutated SCN5A in pigs [Gene-targeted animal models predominantly in mice have greatly expanded our mechanistic understanding of long QT syndrome (LQTS), short QT syndrome (SQTS), Brugada syndrome, catecholaminergic polymorphic ventricular tachyarrhythmia (CPVT), sick sinus syndrome, cardiac conduction disease, and familial AF . Genetic in pigs •. IndeedAnimal models have significantly expanded our understanding of the mechanistic underpinning of arrhythmogenesis, but there exist advantages and limitations that should be considered. Besides the obvious issues with costs and the length of time required for model generation, there are inherent species-dependent differences at the molecular and cellular levels that impact pathophysiology. Indeed, these differences must be evaluated and understood in order to translate therapeutic findings to clinical practice. Advantages of using small animal models are their relatively inexpensive costs, short gestational period and large litter size, and relative ease of genetic manipulation. However, small animals possess cardiovascular anatomy and physiology that are substantially different from humans . For insStudies in cardiac arrhythmogenesis have also taken advantage of the ex vivo Langendorff-perfused heart Fig. . IsolateFor deciphering the mechanisms of arrhythmogenesis, in vitro models are useful tools that can simplify experiments by restricting the factors of interest to be tested independently. The models vary from murine to human cardiomyocytes and from single cells to multi-cellular preparations Fig. . Each of2+ or K+ currents were determined in single cardiomyocytes [Primary cardiomyocytes can be harvested from animal models and are critical for our understanding of mechanisms of arrhythmias at the cellular and subcellular levels. The source of focal arrhythmic activity—spontaneously generated or triggered AP—either due to ion channelopathies, altered ion channel expression, subcellular localization, or post-translational modifications, and drug-modulated channel function, can be readily deciphered using patch-clamp and imaging techniques. Myocardial infarction models exhibiting arrhythmias in vivo that have been attributed to reduced Camyocytes , 62. Undmyocytes . StudiesA readily available source of cardiomyocytes is highly desirable for in vitro studies; however, the lack of regenerative capability of adult cardiomyocytes requires that primary culture is used as the main source. To circumvent limited proliferative potential of cardiomyocytes, cell lines have been generated by restoring the proliferative ability. This can be achieved by infecting cardiomyocytes with simian virus (SV) 40, a type of DNA tumor virus, that expresses large and small T antigens, which cooperatively inhibit tumor suppressors and transform the cells to escape senescence . HL-1 ceConsiderable insights in cardiac arrhythmias have been derived from various species. However, the mechanisms responsible for arrhythmogenesis in the context of human electrophysiology may be significantly different from animal models with differing electrophysiology. Therefore, human cardiomyocytes remain the most reliable model for investigating the mechanisms of human arrhythmias. Primary human cardiomyocytes are not easily obtainable. Their laboratory usage is further limited by the difficulty of maintenance in culture. HiPSCs that can differentiate into all somatic cell types, including cardiomyocytes, have garnered great interest in recent years as a readily available option. These cells not only open the possibility of having an unlimited cell source as well as genetic modification or imposed environmental conditions but hiPSCs can also be generated from patients suffering from cardiac arrhythmias to generate patient-specific in vitro models. To date, numerous LQTS-, SQTS-, Brugada syndrome-, and CPVT-hiPSC lines have been generated to study the channelopathies –73. CardHiPSC-derived cardiomyocytes are not, however, without disadvantages. There are two key issues associated with using these cells for studying arrhythmias: (1) the heterogeneity of cardiomyocyte subtypes in the differentiated population and (2) immaturities of hiPSC-derived cardiomyocytes. Widely used cardiomyogenesis protocols to differentiate hiPSCs yield a heterogeneous population of pacemaker-, atrial-, and ventricular-like subtypes, as classified by the AP profiles . The hetA few strategies have been devised to minimize subtype impurities. Enrichment of desired cardiomyocyte subtype requires a surface marker with high specificity for the subtype. Ventricular cardiomyocyte enrichment by selecting CORIN-positive or CD77-positive/CD200-negative cardiomyocytes has been reported, but the specificity could still be improved , 81. An Immaturity of hiPSC-derived cardiomyocytes compared to adult cells, from excitation to contraction, is another issue that may confound experimental results. For instance, the pacemaking, hyperpolarization-activated cyclic nucleotide-gated (HCN4) channels that are absent in the adult working cardiomyocytes are present in all hiPSC-derived cardiomyocytes . ConversTo remedy this developmental deficiency, maturation strategies have been devised, which range from electrical, mechanical, to metabolic conditioning –95. SignOne arrhythmogenic mechanism that fails to be captured at the single cell level is the impulse propagation in arrhythmia formation. Ectopic beats triggered by automaticity, early or delayed after depolarizations (EADs or DADs) are focal events that only lead to arrhythmia if these are propagated to the neighboring cells. Consequently, arrhythmias can only be observed in multi-cellular tissue models. Besides cellular contribution of abnormal electrophysiology, cell-cell electrical coupling affecting the conduction velocity is another factor that dictates the wave length of the AP wave front. Additionally, electrophysiological heterogeneities, including dispersion of repolarization or source-sink mismatch, are triggers for arrhythmias that only become apparent at the tissue levels.Conduction velocity and wave front reentry can be observed in either 2D or 3D tissue models composed of a syncytium of cardiomyocytes via optical mapping. Since primary adult cardiomyocytes from animals do not form monolayers in culture, most in vitro tissue models have relied on primary neonatal or hiPSC-derived cardiomyocytes that retain morphological plasticity to reorganize and allow electrical coupling with neighboring cells. For the 2D model, cardiomyocytes plated in typical cell cultureware exhibit random cellular orientation unlike the anisotropic alignment of the myocardium. Conduction pattern is affected by the cardiomyocyte alignment, which is reflected in the increased incidence of arrhythmic events even in healthy monolayer of hiPSC-derived cardiomyocytes, making this model less accurate for testing pro-arrhythmic triggers. With advances in microfabrication technology, patterned surface to induce aligned monolayers of hiPSC-derived cardiomyocytes has been shown to reduce the incidence of arrhythmia, creating a more stable baseline tissue model for assessing pro-arrhythmic factors , 97.While 2D hiPSC-cardiomyocyte-based tissue models can display arrhythmic events, there are limitations in the organization and maturity of cardiomyocytes. Specifically, 3D tissues in a patch configuration have been reported to be necessary in modeling arrhythmic phenomenon such as torsade de pointes with fluctuating excitation intervals that can only be recreated in the presence of a meandering wave origin . This is2+ dynamic, APs, and mechanical contractions, multiscale computational modeling offers unparalleled advantages in deciphering cardiac arrhythmia mechanisms (Fig. Since cardiac excitability is sculpted by the beat-to-beat feedback among three nonlinear dynamic processes including Casms Fig. . The funThe early mathematical models of the AP from adult human atrial cells were developed by Nygren et al. and Courtemanche et al. in 1998 , 111. WiThe first mathematical model of the AP from human ventricular myocytes was reported by Priebe and Beuckelmann in 1998 , 127. Th2+ handling [Purkinje fibers play critical roles as initiating sites for ventricular tachycardia and fibrillation. An AP model of human Purkinje fibers was first developed by Tusscher and Panfilov . Based ohandling . The strhandling .2+ clock and constructs a comprehensive mathematical model for SAN cells [Because of the very limited experimental data from human SAN, the development of mathematical model for human SAN lags behind the model development for human atrial and ventricular myocytes. Seemann et al. published the first human SAN AP model as part of a 3D human atria model . This isAN cells .One major advantage of computational modeling is the expansion of personalized medicine by integrating patient information and clinical data into the model to develop personalized treatment strategies . A recenFinally, the innervation, spatial, and temporal dynamic feedback at tissue and organ levels are required to be integrated into the models for understanding the beat-to-beat electrical excitability in the heart. Therefore, joint efforts of computational biologists, experimental biologists, and physicians are necessary to integrate the multi-discipline knowledge in computational modeling. Additional validation with experimental and clinical data followed by reiterative refinement of the computational models will help to pave the way for deeper understandings of the complex mechanisms of cardiac arrhythmogenesis, assist the diagnosis, and guide the treatment.The mechanistic understanding of cardiac arrhythmogenesis is critical for the development of cardiac cell-based therapy. Animal models from multiple species provide experimental platforms for mechanistic hypothesis testing. Ex vivo and in vitro models enable the application of several cutting-edge techniques including the development of patient-specific hiPSC-CMs and CRISPR-Cas9 gene editing. Recent advances in multiscale computational models for arrhythmogenesis represent the joint efforts by computational biologists, biophysicists, experimental biologists, and physicians, and provide quantitative and powerful tools for the mechanistic understanding of arrhythmogenesis in cardiac cell-based therapy. Further integrations of experimental, computational, and the virtual heart model will provide critical insights into novel mitigation strategies for cardiac arrhythmias in cell-based therapy.


Glucocorticoids are steroid hormones with key roles in the regulation of many physiological systems including energy homeostasis and immunity. However, chronic glucocorticoid excess, highlighted in Cushing’s syndrome, is established as being associated with increased cardiovascular disease (CVD) risk. Atherosclerosis is the major cause of CVD, leading to complications including coronary artery disease, myocardial infarction and heart failure. While the associations between glucocorticoid excess and increased prevalence of these complications are well established, the mechanisms underlying the role of glucocorticoids in development of atheroma are unclear. This review aims to better understand the importance of glucocorticoids in atherosclerosis and to dissect their cell-specific effects on key processes . Clinical and pre-clinical studies have shown both athero-protective and pro-atherogenic responses to glucocorticoids, effects dependent upon their multifactorial actions. Evidence indicates regulation of glucocorticoid bioavailability at the vasculature is complex, with local delivery, pre-receptor metabolism, and receptor expression contributing to responses linked to vascular remodelling and inflammation. Further investigations are required to clarify the mechanisms through which endogenous, local glucocorticoid action and systemic glucocorticoid treatment promote/inhibit atherosclerosis. This will provide greater insights into the potential benefit of glucocorticoid targeted approaches in the treatment of cardiovascular disease. Glucocorticoids are steroid hormones with an essential role in the regulation of numerous physiological processes, including energy utilisation, immune response, blood pressure, mood/memory, and stress responses . Their sIn the circulation, the bioavailability of glucocorticoids is regulated by plasma binding proteins—primarily corticosteroid-binding globulin (CBG), but also albumin . Under nGlucocorticoids have important roles in physiology and are key in the maintenance of cardiovascular health. This review will focus on the ability of glucocorticoids to regulate atherosclerosis, both in terms of driving the pathogenesis of the disease (pro-atherogenic), and as a potential therapeutic option to limit disease severity (athero-protective). Here we will outline the historical literature and explore the more recent evidence underlying how glucocorticoids influence the development of atheromatous plaques, in particular dissecting the cell-specific mechanisms regulating glucocorticoid action in this disease setting.The ability of glucocorticoids to regulate numerous key physiological processes, in particular the body’s immune response and fuel metabolism, mean they play an important role in the maintenance of cardiovascular health. However, these essential, adaptive responses can become maladaptive upon chronic exposure to elevated levels of glucocorticoids . Indeed,The most striking demonstration of the cardiovascular consequences of glucocorticoid excess remains that highlighted in the clinical presentation of Cushing’s syndrome . IndividWhilst cases of Cushing’s are extremely rare, affecting between 40 and 70 people out of every million , the linA further link between glucocorticoid excess and the development of cardiovascular disease has been suggested in patients with metabolic syndrome; a condition associated with increased cardiovascular risk . The simIn addition to the evidence obtained from exposure to elevated endogenous glucocorticoids, the widespread therapeutic use of glucocorticoids to treat inflammatory and autoimmune conditions has also provided supporting evidence for their association with CVD. While effective in alleviating symptoms of asthma, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) , glucocoTogether, the evidence for an association between increased exposure to glucocorticoids and increased CVD risk is clear, yet the question of causality remains. Using Mendelian randomisation and data collected across 17 studies of European ancestry, a recent study tested whether genetically elevated cortisol in the general population is causally associated with a number of cardiovascular risk factors . ImportaAtherosclerosis is a progressive inflammatory disease, characterised by a narrowing of the arteries due to vascular remodelling and formation of atheromatous plaques . The cliIn line with our current understanding from pre-clinical models, there are two key events in atherogenesis: endothelial cell damage and vascular remodelling. In particular, the recruitment and activation of circulating immune cells, lipoprotein retention, and the migration and proliferation of vascular smooth muscle cells (vSMCs) are viewed as critical cellular responses. Research has established that initial intimal thickening of arteries is physiological, developing silently throughout an individual’s lifetime, and is evident in young children and even babies less than a year old . HoweverThe proliferation and migration of vSMCs from the media to the intima forms another significant component of vascular remodelling. Under the pro-inflammatory environment, vSMCs undergo a phenotypic switch in which they lose their contractile phenotype, releasing growth factors and matrix metalloproteases (MMPs), and gain a more macrophage and foam cell-like phenotype ,43. As tA major aspect of vascular remodelling is the degradation of extracellular matrix (ECM), which occurs as a consequence of protease action. Neutrophil elastase (NE) and MMPs, released from activated neutrophils, macrophages, endothelial cells and vSMCs are key proteases that promote the breakdown of ECM by degrading structural proteins such as collagen and elastin ,45. The −/− mice can have features of vulnerability, but death caused by plaque rupture is not evident [In humans, most plaques are stable and remain asymptomatic; however, plaques can rupture resulting in thrombosis and occlusion of arteries. The “stabilisation” of plaques is regarded as any change in the composition in which reduces the plaques vulnerability to rupture or erode. Therefore, identifying characteristics of these “unstable” plaques is crucial. Indeed, stable plaques have thick protective fibrous caps with small necrotic cores with less macrophage infiltration . The que evident .Additionally, it is important to recognise that approximately 20% of deaths in acute coronary syndrome (ACS) patients can be caused by superficial plaque erosion, which has a distinct mechanism from plaque rupture . TypicalThe cumulative evidence gathered over the past 20 years highlighting increased atherosclerosis in Cushing’s patients suggests that glucocorticoids may be pathophysiological drivers of plaque development. Individuals with Cushing’s exhibit higher circulating LDL levels, a thickened intimal-medial layer, and a more narrowed lumen in the carotid artery compared to age- and sex-matched non-Cushing’s individuals . A more The lesion burden of lower limb PVD patients is greater in individuals that have had long term (>5 years) glucocorticoid treatment compared to individuals that have plaques but no previous exposure to exogenous glucocorticoid . In partAlthough chronic, supraphysiological exposure to glucocorticoids increases the risk and burden of atherosclerosis in humans, the earliest evidence of glucocorticoid action in pre-clinical models indicated a protective role. In cholesterol-fed rabbits, co-administration of “low dose” dexamethasone (0.125 mg/day) protected against further progression of atherosclerosis, with reduced plaque development, macrophage recruitment and subsequent foam cell formation ,63. In m−/− mice, a chronic increase in endogenous corticosterone levels in response to repeated stressful stimuli increased the prevalence of lesions compared to unstressed mice [−/− mice, compared to controls [−/− background compared to controls [In contrast, a number of pre-clinical studies employing manipulations in endogenous glucocorticoid levels, often subtle in comparison to the pharmacological administration seen above, have demonstrated pro-atherogenic effects of glucocorticoids. In ApoEsed mice . Similarcontrols , while mcontrols . Intrigucontrols ,69.From an overview standpoint there appears compelling evidence for glucocorticoids to act in both an adverse and beneficial manner in the context of atherosclerosis. However, the question of whether glucocorticoids can be viewed as drivers of atherosclerosis or anti-atherogenic is more likely too simplistic an approach. Rather, it is becoming clear that the key to understanding this apparent paradox requires a multifactorial approach, and lies in identifying the crucial cellular targets of glucocorticoids in the vasculature compared to the broad targets resulting from systemic administration, and in parallel understanding the mechanisms that such cells utilise to regulate their response to glucocorticoids.The importance of glucocorticoids in maintaining vascular reactivity is well established, and they are vital for regulation of blood pressure and blood flow ,71. HoweA and ETB receptors in a paracrine manner on vSMCs to cause contraction, and in an autocrine manner on endothelial cells to cause relaxation [2+-mediated contraction [Glucocorticoids can influence vascular reactivity through modulating either vasoconstriction or vasodilation . Endothelaxation . In humalaxation ,75. Earllaxation ,77, an elaxation . The prolaxation . Howeverlaxation . These ftraction . Moreovetraction .2) from endothelial cells [Glucocorticoids can also regulate vascular tone through modulation of vasodilation. In particular, they can inhibit the synthesis and release of the vasodilators nitric oxide (NO) and prostacyclin  enzymes has been extensively reviewed ,109,110.−/− mice reduced neointimal thickening and increased collagen content, promoting plaque stability [−/− mice demonstrated similar protection from atherosclerosis [Pre-clinical studies using genetic deletion and pharmacological inhibition of 11β-HSD1 implicate this enzyme as a driver of atherosclerosis . The inhtability . In a sitability . Two indclerosis ,69. In tclerosis . This maclerosis . Thus, t−/− mice were crossed with 11β-HSD2−/− mice. These mice exhibited increased development of severe atheromatous plaques even in that absence of high-fat/Western diet, a mechanism attributed to glucocorticoid-mediated activation of MR [The majority of glucocorticoid effects are a consequence of transcriptional actions mediated through intracellular binding to either the low-affinity GR or the high-affinity MR ,8,9. Upoon of MR . In a seon of MR . Interes−/− model of atherosclerosis [−/− mouse model of atherosclerosis did not alter lesion size or macrophage content [In contrast to endothelial MR activation appearing to promote atherosclerosis, evidence indicates an athero-protective role for GR activation. Endothelial cell-specific GR deletion results in more severe atherosclerosis and increased recruitment of macrophages in an ApoEclerosis . However content . InteresSERPINA6/1 locus influencing hepatic SERPINA6 expression associated with morning plasma cortisol [Under physiological conditions, CBG binds 80–90% of circulating glucocorticoids, with 2–10% remaining unbound. Consequently, in line with the free hormone hypothesis, this means that only between 2 and 10% of glucocorticoids are able to diffuse into tissues and cells and exert biological activity. Recent genome-wide association studies have demonstrated the importance of CBG in regulating circulating levels, with genetic variation in the cortisol ,124.Unsurprisingly, given the role of glucocorticoids in numerous physiological settings, CBG has been postulated to play a significant role in glucocorticoid action in several pathological conditions. For example, in sepsis, a reduction in plasma CBG levels gives rise to an increased free fraction of circulating cortisol in the circulation ; an impoHowever, it is not only alterations in the levels of CBG which can influence glucocorticoid bioavailability. Structural changes in CBG protein are known to regulate its affinity for glucocorticoids. In particular, proteolytic cleavage of the reactive certain loop of CBG is known to result in an approximately 10-fold decrease in its binding affinity for cortisol ,131. A sWhile it is clear that chronic, systemic glucocorticoid excess is positively associated with increased atherosclerosis, evidence is now being uncovered for a causative role for glucocorticoids in the cardiovascular complications arising from atherosclerotic plaque formation. This has re-ignited the debate around the influence of glucocorticoids in disease progression and treatment. The ability of glucocorticoids to affect multiple cell types within the vasculature, in parallel with mediating opposing actions depending on the concentration and cellular target, makes determination of relative importance difficult. The situation is made more complex by the multiple regulatory mechanisms still being discovered that control glucocorticoid bioavailability, both at sites of plaque development and in specific cells. Pre-clinical studies in genetically modified mice continue to aid our understanding of these mechanisms, identifying potential new therapeutic targets to control glucocorticoid action. However, it is clear that while these mechanisms can alter glucocorticoid action, such changes need to be considered not only in the context of specific cells, but in the context of plaque site, disease progression and treatment. For example, limiting vSMC migration may be viewed as beneficial in limiting necrotic core formation, but it may lead to a fibrous cap more vulnerable to rupture. Given the widespread use of animal models, a greater understanding of the species-specific differences in plaque composition and vulnerability would also be of significant benefit. Together with a better knowledge of the mechanisms controlling local, endogenous glucocorticoid action, these insights will be key in both predicting the response of patients to systemic glucocorticoid treatment, and in advancing personalised, targeted glucocorticoid therapy to combat cardiovascular disease.


The pathogenesis of Acute Rheumatic Fever/Rheumatic Heart Disease (ARF/RHD) and associated neurobehavioral complications including Sydenham's chorea (SC) is complex. Disease complications triggered by Group A streptococcal (GAS) infection are confined to human and determining the early events leading to pathology requires a robust animal model that reflects the hallmark features of the disease. However, modeling these conditions in a laboratory animal, of a uniquely human disease is challenging. Animal models including cattle, sheep, pig, dog, cat, guinea pigs rats and mice have been used extensively to dissect molecular mechanisms of the autoimmune inflammatory responses in ARF/RHD. Despite the characteristic limitations of some animal models, several rodent models have significantly contributed to better understanding of the fundamental mechanisms underpinning features of ARF/RHD. In the Lewis rat autoimmune valvulitis model the development of myocarditis and valvulitis with the infiltration of mononuclear cells along with generation of antibodies that cross-react with cardiac tissue proteins following exposure to GAS antigens were found to be similar to ARF/RHD. We have recently shown that Lewis rats injected with recombinant GAS antigens simultaneously developed cardiac and neurobehavioral changes. Since ARF/RHD is multifactorial in origin, an animal model which exhibit the characteristics of several of the cardinal diagnostic criteria observed in ARF/RHD, would be advantageous to determine the early immune responses to facilitate biomarker discovery as well as provide a suitable model to evaluate treatment options, safety and efficacy of vaccine candidates. This review focuses on some of the common small animals and their advantages and limitations. The concept of comparative medicine developed based on the theory that animal species share physiological, anatomical and behavioral characteristics similar to human . This coPost-streptococcal autoimmune disorders are complex immune mediated disease mostly affecting children and young adults following exposure to Group A streptococcal (GAS) infection. These includes ARF, RHD, Sydenham Chorea (SC) and possibly, pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS) –5. AfterVariety of host, bacterial, socioeconomic and environmental factors contribute to the prevalence and incidence of ARF/RHD . EnvironThe pathophysiology of post-streptococcal complication is not fully understood, however antigenic mimicry between GAS antigens and host proteins is partly considered as factor that triggers autoimmunity. It may also be affected by several environmental, genetic and socioeconomic factors. Although an autoimmune process has long been considered to be responsible for the initiation of ARF/RHD, it is only in the last few decades that the mechanisms involved in the pathogenesis of this post streptococcal conditions have been unraveled partly due to experimentation on animal models. Studies have shown that molecular mimicry of streptococcal antigens enable the generation of antibodies that bind to both GAS antigens and cross-react with host tissue proteins including cardiac myosin, collagen I and IV, tropomycin, laminin, vimentin, and keratin .Further studies have demonstrated that human collagen IV, one of the major components of the basal membrane, a layer of extracellular matrix secreted by epithelial cells, can also be involved in the pathogenesis of ARF/RHD by acting as an autoantigen after forming a complex with GAS antigens . SeveralPost streptococcal autoimmune complications including ARF/RHD is uniquely a human condition and humans are the only host and reservoir for GAS. Thus, modeling post-streptococcal autoimmune complications in animal is challenging. However, animals are the only experimental models used to investigate the characteristic signs, pathogenesis and pathophysiology specific to ARF/RHD. Animals including guinea pigs, rabbits, pigs, sheep, goats, cattle, cats, dogs, and non-human primates have been used as experimental model to understand the disease mechanism of ARF/RHD and to investigate the rheumatogenic potential of GAS M proteins –36. In tThe early experiments on rheumatic myocarditis were carried out in rabbits based on the hypothesis that ARF/RHD was caused either by direct streptococcal infection or by direct damage to heart tissues by streptococcal toxins. However, none of the rabbits showed similar pathology to rheumatic myocarditis in these studies . A studyMacaca mulatta) induced typical RHD lesions as well as evidence of myocarditis and valvulitis along with infiltration of lymphocytes, histiocytes, Anitshkow cells, and plasma cells  . Quinn eLewis rats immunized with recombinant M6 (rM6) protein demonstrated valvulitis and focal myocarditis, which were histologically similar to pathological lesions observed in patients with RHD . Later sStreptococcus dysgalactiae subsp. equisimilis (SDSE/GGS), Lewis rats developed typical histological lesions with infiltration of inflammatory cells into cardiac tissue providing experimental evidence that streptococci other than GAS could trigger and/or exacerbate post-streptococcal carditis  and pediatric autoimmune neuropsychiatric disorders associated with streptococcus (PANDAS) . SC is aIn the past many studies have been carried out to develop an animal model to investigate the post streptococcal neurobehavioral disorders  57–62).–62.57–62in vitro studies demonstrated that monoclonal antibodies against N-acetyl-β-D-glucosamine and lysoganglioside GM1 induced the activity of calcium/calmodulin-dependent protein kinase II, which is potentially implicated as an important mediator of learning and behavior (t+/GFP and RORγtGFP/GFP mice) have shown reduced BBB leakage, microglial activation, and antibody infiltration into the brain following intranasal challenge with GAS in vitro with GAS . This dereplacement, reduction and refinement) determining different aspects of a disease in a single animal will minimize the number of animals needed for research (Post streptococcal autoimmune sequelae is a multisystem disorder affecting multiple organs including heart, brain, joints, connective tissues and skin . The immresearch . Recentlresearch . Moreoveresearch , 73–75, research . To furtWhile significant advances in animal models of ARF/RHD have been made in the last decade, there is still a paucity in pre-clinical studies on other complications associated with ARF/RHD including neurobehavioral changes, arthritis and skin manifestations. Arthritis is observed in ~50–70% of patients with ARF and is a major Jones Criterion for the diagnosis of ARF. However, none of the animal studies have investigated GAS induced autoimmune process in subcutaneous tissue and joint tissue in any of these models.Laboratory models are important to determine the early events leading to chronic disease. In particular when clinical studies are not possible during the early stages. In a credible animal model symptoms of physiological, anatomical and behavioral conditions must be comparable to those observed in human disease. In addition, an animal model should be reliable and the changes observed must be reproducible across laboratories. An animal model of ARF/RHD and associated neurobehavioral complications should possess functional and pathological changes encompassing motor deficits as well as compulsive and stereotyped behaviors similar to SC. Since genotypes, sex and age difference affects the development of autoimmune complication; selection of an appropriate animal model is important to investigate the pathogenesis of ARF/RHD and associated complications. Several animal models have been tested to investigate the onset, and progression of ARF/RHD. The Lewis rat model characterized by us and others, is a reliable model to investigate early events that lead to cardiac valvular pathology. Together with advances in novel imaging technologies and integrated computational approaches our model will provide the means to address these challenges . ImportaRAMR, SS, ASH, NMA, KSS, DJM, and NK wrote the main manuscript. All authors have read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pesticides have been used extensively in the field of plant protection to maximize crop yields. However, the long-term, unmanaged application of pesticides has posed severe challenges such as pesticide resistance, environmental contamination, risk in human health, soil degradation, and other important global issues. Recently, the combination of nanotechnology with plant protection strategies has offered new perspectives to mitigate these global issues, which has promoted a rapid development of NCs-based pesticides. Unlike certain conventional pesticides that have been applied inefficiently and lacked targeted control, pesticides delivered by nanocarriers (NCs) have optimized formulations, controlled release rate, and minimized or site-specific application. They are receiving increasing attention and are considered as an important part in sustainable and smart agriculture. This review discussed the limitation of traditional pesticides or conventional application mode, focused on the sustainable features of NCs-based pesticides such as improved formulation, enhanced stability under harsh condition, and controlled release/degradation. The perspectives of NCs-based pesticides and their risk assessment were also suggested in this view for a better use of NCs-based pesticides to facilitate sustainable, smart agriculture in the future. To feed nearly 10 billion people by 2050, food production would need to increase at least 50% from the 2012 level . UnfortuThe long-term unmanaged use of pesticides might be reversed by a new-emerging agri-tech revolution called ‘smart agriculture’ that is based on nanotechnology and aimed at providing agricultural supply for food and industry in an efficient and sustainable manner Equipped with a comprehensive farming system that covers multiple, continuously updated technologies, the ‘smart plant’ in smart agriculture can be edited to report its real-time requirements . SubsequMost current active ingredients (AIs) of pesticides are commonly hydrophobic compounds with poor water solubility, resulting in the excessive use of both AIs and harmful organic solvents to ensure their dispersion in formulations and subsequent pesticidal effect . NanotecApart from formulation optimization, targeted application of pesticides is also smart agriculture to ensure pest control effect and reduce toxicity to nontargets. RNA interference (RNAi) mediated pest control has long been a leading part of targeted application of pesticides ,25,26. TAedes aegypti  and Chilosuppressalis (a destructive rice pest in China). A successful application of dsRNA delivery system to control pests was shown in Blattella germanica . . BacilluEucalyptus citriodora were made into nanoliposomes to fight against Staphylococcus aureus [Achillea millefolium L.) [Tetranychus urticae Koch in fumigant experiments and contact lethality tests. The essential oils delivery system based on NCs is another successful application. As a promising biocontrol sources, the plant essential oils also have a high pesticidal activity, a low mammalian toxicity but a strong volatility, leading to a lack of persistence . Two or s aureus . This anlium L.) . This EOA continuous release of Emamectin-benzoate over 200 h was also observed after an encapsulating it into microspheres composed of polymeric stabilizer polyvinyl alcohol and non-ionic surfactant polyoxyethylene castor oils. The microsphere also had excellent antiphotolysis performance and good leaf distribution . Theses Chemical AIs with unsatisfying formulation undergo a premature emission into the environment, resulting in short-lived control effects of pests and a toxic risk to nontargets. These disadvantages have been reversed through a NCs-based bifunctional delivery systems that offer protection and slow release of AIs. In these systems, mesoporous nanomaterials (MNs) such as nanoclay, activated carbon, and porous hollow silica are widely used as protective substrates . They ofPlutellaxylostella [Myzus persicae [As discussed above, more and more advanced NCs have been applied in delivery systems to enable pesticides with good protection, sustained release, low toxicity, and other excellent properties. In advanced smart systems, NCs was grafted with stimulation-responsive groups to facilitate a regulated release of AIs by stimulus conditions such as pH, thermal, light and enzyme of pests, which can not only control pests effectively but also reduce environmental contamination ,71. Theslostella . Anotherpersicae . The alkaline-response oral delivery systems play a role in targeted control of insect pests by triggering the release of insecticides in alkaline guts. Kumar et al. fabricated pH-dependent release system using two natural materials of alginate and chitosan, via an ionic pregelation and a polyelectrolyte complexation route . The rel2 photocatalysts also contributed a photocatalytic oxidation degradation of abamectin pesticides after a temporal threshold for pest control [Recently, light-responsive delivery systems have gained considerable interest. A light-controlled delivery system was well developed by conjugating photolabile 2-nitrobenzyl with carboxymethyl chitosan to deliver herbicides  . This sy control . These sThe rapid development of nanotechnology brings new opportunities and driving forces for the agri-tech revolution . PreviouDespite all this progress, the current development of NCs-delivered pesticides is still at an early stage. There is insufficient information about their impact on human health and ecosystem safety. Both NCs-delivered pesticides and other pesticides at nanoscale (defied as nanopesticides) has been a growing concern for the public due to their toxicological risk assessment. Meanwhile, a unified assessment criterion is also needed before commercialization . The EurAnother strategy to reduced toxicity concern is using green materials or green synthesize for NCs production. As far as we know, the frequently reported NCs such as MOFs, adsorption resin, activated clay, activated alumina, and activated carbon have a nondegradable nature even though they have excellent delivery effects ,92. NondAcalypha indica), bacteria (psychrophilic bacteria), yeast (Humicola sp.), and fungi (Aspergillus terreus) [Thus, it is necessary to use relatively nontoxic materials or green methods to synthesize or modify NCs. Biosynthesis, based on living organism or its extracts, is an important part of green technology. The waster cyanobacteria, with both biocompatibility and microalgae surface for drug-loading modification, have reported to be modified as NCs for delivery and controlled release of avermectin . The bioterreus) ,99,100. All the processes of physical, chemical as well as biological methods are being developed to meet certain requirements of NCs synthesis. Chemical synthesis is most effective means to produce NCs and has the most potential to be scaled up for large-scale production. The development of biosynthesis and utilization of physical methods could be alternatives for chemical methods, in an eco-friendly manner and thus reducing the reliance on the toxic chemicals. However, each of the existing protocols suffers from their intrinsic defects. Thus, it is still an open challenge to combine all the methods to produce NCs with less side effect on an industrial scale. A possible future work is presented in NCs-delivered pesticides are giving a fresh insight into sustained agriculture for a greater control efficacy and fewer side effects. It is increasing clear that the smart nanodelivery systems developed in past, present and further will plays vital role in dealing with the worldwide issues like the energy crisis, food contaminations, and deterioration of soils. Solving these issues will require strong collaboration among agronomists, nanotechnologists and informationist, to develop and apply multi-functional NCs-delivered pesticides that are beyond the properties of targeted control and regulated release. The responsibility of farmers could be to find the right time to degrade a smart pesticide by a balance between the high effect and less residues.


Polymeric biomaterials exhibit excellent physicochemical characteristics as a scaffold for cell and tissue engineering applications. Chemical modification of the polymers has been the primary mode of functionalization to enhance biocompatibility and regulate cellular behaviors such as cell adhesion, proliferation, differentiation, and maturation. Due to the complexity of the in vivo cellular microenvironments, however, chemical functionalization alone is usually insufficient to develop functionally mature cells/tissues. Therefore, the multifunctional polymeric scaffolds that enable electrical, mechanical, and/or magnetic stimulation to the cells, have gained research interest in the past decade. Such multifunctional scaffolds are often combined with exogenous stimuli to further enhance the tissue and cell behaviors by dynamically controlling the microenvironments of the cells. Significantly improved cell proliferation and differentiation, as well as tissue functionalities, are frequently observed by applying extrinsic physical stimuli on functional polymeric scaffold systems. In this regard, the present paper discusses the current state-of-the-art functionalized polymeric scaffolds, with an emphasis on electrospun fibers, that modulate the physical cell niche to direct cellular behaviors and subsequent functional tissue development. We will also highlight the incorporation of the extrinsic stimuli to augment or activate the functionalized polymeric scaffold system to dynamically stimulate the cells. Over the past decade, advances in polymer science and engineering have led the progress of the tissue engineering field by providing solutions for innovative materials/structures to guide cellular behaviors. Typical tissue engineering strategies utilize scaffolds as a synthetic alternative for the natural extracellular matrix (ECM) to temporally support the cells, which require a 3D microenvironment resembling the in vivo conditions to develop a tissue with an appropriate structure and function. Polymers, both naturally derived and synthetic, have gained increased interest in the structural materials of tissue engineering scaffolds due to many advantages. These include the broad spectrum of biocompatible polymeric materials that can be used as tissue and cell culture platforms, the flexibility of the polymers that can be fabricated into various shapes with desired morphological features such as pores and their interconnectivity conducive to cell in-growth, and the existing mature synthesis technologies that enable the polymeric scaffolds to be easily and reproducibly produced. Hydrogel is the most commonly used polymeric biomaterials in tissue engineering due to its unique structural similarities to the native ECM .Hydrogel is a 3D network of either physically or chemically cross-linked polymer chains that hold a large number of water molecules. Such a flexible structure of the hydrogel yields its control over shape, porosity, and surface morphology, providing a versatile platform for tissue engineering applications, including cell culture scaffolds, tissue barriers, and drug delivery vehicles . More reWhen a high-voltage electric field is applied between a polymer droplet and a collector, the polymer droplet forms a cone shape, known as the Taylor cone, that ejects a jet of the polymer solution. The electrostatic repulsion and the rapid solvent evaporation will then separate the solution and create nano- or microfibers, which are attracted to and deposited onto the collector. Such fibrous structure formed by electrospinning resembles native ECM, supporting cell growth, differentiation, and maturation .Besides appropriate structural support, there are several properties that need to be taken into consideration when designing polymeric tissue scaffolds, including; (1) low cytotoxicity of polymers and their breakdown products; (2) good biocompatibility with low immunogenicity to reduce inflammatory responses after the implantation; (3) an appropriate rate of biodegradability designed for a specific tissue and its anatomical location; (4) high cell adhesion properties for the tissue morphogenesis of adherent cell types; (5) capability to provide appropriate chemical and physical microenvironment to the cells. To meet all these requirements, especially furnishing an adequate microenvironment for the cells, the scaffolds need to provide more than simple structural support by presenting various physicochemical cell niches. The most common and well-studied method is chemical functionalization, including polymer surface modification and biochemical delivery .Surface coating can be easily achieved by physical adsorption or chemical conjugation of functional molecules to various natural and synthetic polymers, such as chitosan, collagen, polyvinylidene fluoride (PVDF), poly(ε-caprolactone) (PCL), and poly(L-lactic acid) (PLLA) ,6,7. SurDue to the complexity of cellular microenvironments in the native tissues, however, chemical modification is usually insufficient to fully develop functionalized tissues in vitro. In this regard, the control over physical microenvironments, including electrical, mechanical, and magnetic factors, has gained significant interest since they have been recently shown to crucially influence cellular behaviors, such as migration, proliferation, differentiation, and maturation, as well as to enhance tissue regeneration in bone, nerve, and blood vessels . Unlike In recent years, many excellent review articles discussed various aspects of polymeric scaffolds including synthesis, structuring, chemical modification as well as their clinical applications ,24,25,26Electrical signals are ubiquitous in the physiological system where endogenous electric fields play a vital role in biological processes ranging from early embryonic development to tissue regeneration ,29,30,31It was also demonstrated how electric cues enhanced various regenerative cellular activities such as neurite outgrowth in nerve cells and enhanced collagen production and calcification in bone cells . Based oElectrically conductive polymers (CPs) are a class of novel materials that enable the direct application of electrical and electrochemical stimuli to tissues and cells , as listCrowder et al. demonstrated the functionality of an electrospun PCL scaffold embedded with CNTs to improve the cardiac differentiation of MSCs , which eMoreover, Kharaziha et al. fabricated hard and flexible hybrid CNT-containing poly (glycerol sebacate)/Gelatin nanofibrous scaffolds with improved electrical properties which facilitated better beating action from cardiomyocytes . The gelDue to their unique electrical properties, polypyrrole (PPy), polyaniline (PANi), and poly (PEDOT) are the common standalone CPs that are frequently utilized in the field of tissue engineering ,65. PPy For neural tissues, Huang et al. fabricated a biodegradable conductive composite of PPy and chitosan to apply external electrical stimulation to Schwann cells, which revealed that low voltages (100 mV/mm) induce beneficial effects on cellular activities but higher voltages (300–1000 mV/mm) cause detrimental effects. Neurite outgrowth was also shown to be highly elevated by electrical stimulation applied through the conductive scaffold in vivo; the production of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) from Schwann cells was considerably elevated by electrical stimulation, which might have contributed to enhanced neurite outgrowth and nerve regeneration .Another research group seeded osteoblasts-like Saos-2 cells on an electroactive layer made of PLA and bioactivated PPy using heparin (PPy/HE) . The effPANi is another CP that offers ease of synthesis, biocompatibility, low cost, as well as natural antibacterial properties . PANi isPEDOT has been considered as an alternative to PPy due to its greater resistance to oxidation and higher conductivity. In vitro toxicity and biocompatibility tests have shown that PEDOT is cytocompatible ,72. PEDOThe potential of conductive polymers in tissue engineering is significant because the electrical regulation of cellular activities is essential for the regeneration of injured tissues. However, there are certain obstacles when CPs are employed in tissue engineering. The glaring shortcomings of the available systems are poor polymer–cell interactions, relatively low biocompatibility of by-products, poor solubility, and processability, as well as independently uncontrollable mechanical properties. The inability of CPs to degrade at an appropriate rate is one of the greatest constraints for tissue engineering usages. In vivo persistence of CPs for a long time may trigger inflammatory reactions and the requirement for a second surgical process. The synthesis of materials with both electroactive and degradable attributes is extremely desirable which, however, remains a challenge. There are ongoing efforts to address such a challenge by new materials and different synthesis methods for obtaining scaffolds that are both biodegradable and electrically conductive ,65.Tissues and cells in vivo constantly experience evolving mechanical microenvironments depending on the anatomical location and their developmental stage. Numerous studies have found that physical cues, including morphology, topography, availability of adhesion sites, and mechanical properties of substrates, play a crucial role in cellular behaviors ,76,77,78Mechanically tuned scaffolds can provide a platform to intrinsically  or extrinsically  control mechanical environments to achieve desired cellular responses, as listed in Hydrogels are commonly utilized biomaterials to investigate the mechanotransduction behaviors of tissues and cells due to their characteristics of good biocompatibility, effective mass transfer , similarFurthermore, Rammensee et al. synthesized bis-acrylamide/oligonucleotide polyacrylamide (PAM) hydrogels whose stiffness could be reversibly regulated by controlling the number of DNA hybridization crosslinks . NSCs exFibrous scaffolds synthesized by electrospinning have also been widely explored to guide cell proliferation and differentiation since nanofibrous morphology mimics the structure of the native ECM . DependiOur research group previously explored the relationship between the mechanical properties of electrospun fibrous substrates and induced pluripotent stem cells (iPSCs) colony morphology . The resBesides the method of utilizing different polymer materials to control the stiffness of the cell culture scaffolds , core–shVarious nanofibers composed of different polymer precursors such as PCL, PES, polycarbonate-urethane, or polyether-ketone-ketone (PEKK) were utilized to examine the relationship between substrate stiffness and the differentiation behavior of iPSCs . The resBesides the effects of the intrinsic mechanical properties of polymeric scaffolds on tissues and cells, extrinsic mechanical control of tissues and cells has become a promising method to modulate biological responses. The application of external stress or strain requires a scaffold with suitable physical properties such as stiffness and morphology, which also influence cell fate through the activation of different cell signaling pathways. Unlike tensile forces which can be applied to adherent cells on any flexible substrate with a proper surface modification, the application of compressive forces requires a scaffold that provides a 3D microenvironment for appropriate cell viability, proliferation, and differentiation while transferring applied forces to the cells.The hydrogel system has been the most common platform for such studies as it provides in vivo like microenvironments by encapsulating the cells in a 3D space. Compressive strains with a physiologically relevant magnitude on stem cells encapsulated within agarose or PEG hydrogels have been shown to induce chondrogenesis of stem cells while their effectiveness depended on the degree of lineage specification ,105. KooIn addition, Steinmetz et al. developed a hydrogel system having separate layers of different stiffness and demonstrated a compressive strain-dependent MSC fate specification where high compressive strain enhanced chondrogenesis while low compressive loading enhanced osteogenesis a,b 106]106]. As Electrospun scaffolds have also been utilized to investigate the effects of applied compressive forces on cellular behaviors. Typical nano-sized electrospun fibers, however, prevent the infiltration of cells into the 3D scaffold, limiting its application in mechanobiology studies. Among various approaches to overcome the limitation, the use of electrospun microfibrous scaffolds provides a means to enable cellular infiltration throughout the appreciable thickness of 3D scaffolds while maintaining mechanical integrities under applied compressive forces. Using the electrospun microfibrous scaffold, externally applied compressive forces have been shown to induce functional maturation in osteoblasts, enhancing ECM secretion by activating SMAD 1/5/8 phosphorylation through type 1 BMP receptor . AnotherMechanically tuned scaffolds have extensive applications in tissue engineering and regenerative medicine. Numerous in vitro studies showed the great potentials of mechanically tuned scaffolds in directing cellular behaviors, especially guiding stem cell differentiation. However, in vivo studies on mechano-modulation by functional scaffolds are still limited and are of prime importance to exploit their therapeutic potential. Furthermore, the incorporation of other modifications such as biochemical cues into the mechanical control would provide a more robust control over cellular behaviors. Such mechano-stimulatory approaches need to base on a fundamental understanding of the mechanisms of mechano-transduction for the development of tissue-specific scaffolds.The application of magnetic fields is another method to modulate cellular behaviors to aid in tissue formation and wound healing. It has been reported that a magnetic field (MF) and/or an electromagnetic field (EMF) play essential roles in determining cell adhesion, migration, and differentiation, thus affecting tissue regeneration and repair . Specifi2+ and the activation of calmodulin, which are important factors associated with cell differentiation 153]. In This study demonstrates that the activation of piezoelectric scaffolds by exogeneous mechanical stimulation leads to more significant prolonged effects on tissues and cells as compared to static piezoelectric cell culture platforms. The development of highly efficient piezoelectric materials and/or activation methods applicable for in vivo applications will further the application of piezoelectric scaffolds in various tissue engineering fields including bone regeneration, wound healing, and angiogenesis.In addition to their role in structural support, polymeric scaffolds with a variety of physical functionality have gained significant research interest in the past decade to regulate cellular behaviors and direct tissue functions in vitro by controlling the physical microenvironments of the cells. The recent development of multifunctional polymeric scaffolds, in combination with exogenously applied stimuli including electrical, mechanical, and magnetic stimulation, has provided novel tools to guide tissue morphogenesis such as the development of the functional neuronal network, effective bone regeneration, and blood vessel formation.These promising results are expected to lead to the development of functionally mature engineered tissues in vitro for tissue repair implantation, drug discovery platforms, or other diagnostic applications. Such multifunctional polymeric scaffolds have demonstrated anabolic effects on the functional development of tissues and cells. However, there is still a lack of systematic evaluation and control of those physical factors to precisely direct cellular behaviors, resulting in inconsistent or even contradictory results among the studies. Therefore, a more systematic approach needs to be taken to fully understand the effects of various parameters, including magnitude, duration, and frequency of each physical factor, on cell/tissue development. Nevertheless, recent advances in multifunctional polymeric scaffolds are expected to pave the way for efficient tissue engineering strategies for clinical applications.


Drosophila, C. elegan and shellfish  studied since 1957  and fluorescence microscopy to functionally identify and characterize phagocytes from the scleractinian coral, Pocillopora damicornis, and the actiniarian sea anemone, Nematostella vectensis. Their study demonstrated that phagocytes isolated from diverse anthozoan species broadly recognize and engulf targets displaying a range of microbe-associated molecular patterns (MAMPs) and danger-associated molecular patterns (DAMPs), including bacterial and fungal antigens, heat stressed self-cells, and microplastic beads. This foundational knowledge will facilitate more nuanced characterizations of immune responses in coral ecosystems sensitive to environmental perturbation. Tracey et al. demonstrated the value of understanding cellular immunity in cnidarians by using amoebocyte concentrations to monitor cellular immunity in the Caribbean sea fan octocoral, Gorgonia ventalina, susceptible to infection and parasitism from fungus and copepods. Amoebocyte density was used to examine immune related effects associated with environmental parameters, population demography, and host-parasite interactions. Tracey et al. found environment-specific factors and sea fan demography to be key determinants of sea fan immunity and disease amidst fungal and copepod co-infection. These two studies illustrate the importance of foundational studies, that identify and characterize components of early metazoan immunity, in addressing significant and pressing ecological questions.Phagocytosis is a fundamental component of cellular innate immunity. The identification of specialized phagocytic immune cells in early diverging lineages, including coral, remain elusive despite the discovery of phagocytosis in the late 1800s by Élie Metschnikoff,  and DrosEmery et al. and Schmittmann et al. Both studies showed unexpected redundancy and expansions among the evolutionarily conserved immune signaling mechanisms. Since these organisms rely solely on innate immunity to defend against pathogens, these receptor families likely play a key role in recognizing DAMPs and activating host defenses. Emery et al. identified different subphyla of Cnidaria with unique immune PRR repertoires. Emery et al. found that these unique repertoires correlated with life history traits. In sessile, colonial species, that maintained algal symbiosis expanded PRR repertoires were identified in comparison to mobile, solitary, and non-symbiotic counterparts. Schmittmann et al. found that individual sponge genotypes had unique immune gene expression profiles in response to an immune stimulus. In addition to characterizing sponge immune response involving signaling and recognition, including GTPases and post-translational regulation mechanisms like ubiquitination and phosphorylation, they were also able to connect immunity to traits such as holobiont fitness and susceptibility to stress. These studies nicely articulate the connection between immunological complexity and organismal traits that could affect survival in rapidly changing ecosystems. This theme of complexity in immune response compliments other papers in the series looking at immunity from an ecological perspective in co-infection of octocorals by Tracy et al., down to discrete cellular activity level in scleractinian corals and anemones by Snyder et al.Immunological complexity was a common theme within our Research Topic. For example, pathogen recognition receptors (PRRs) were identified in both Our Research Topic section in Frontiers in Immunology also presented novel areas of research in comparative immunology leading to many new and interesting questions for fruitful future investigation. For example, the immunological repertoire of other early diverging metazoans, such as ctenophores and placozoans, remain essentially unknown. Do we see the same type and range of nuanced complexity as the picture beginning to emerge from contemporary studies in Cnidaria and Porifera? In Anthozoa, how does the diverse assemblage of PRRs interact with various DAMPS? And likewise, how specific is the Anthozoa immune response tailored to a perceived pathogen? Building on current research - how are the immune systems of these organisms affected by anthropogenic climate change both regarding immediate plasticity and/or constitutive phylogenetic immune abilities that ultimately affect survival? Are we seeing functional limits in the face of quickly changing environments? How do various biological traits and environmental factors affect immunity, such as growth rate, relative reproductive state, population density, and location ? These and related questions represent exciting emergent areas of research that stem from this Research Topic. The topics covered in this issue have begun to functionally address fundamental questions in invertebrate comparative immunology that we hope will lead to a shift in our understanding of early diverging metazoan immunity and implications in highly unpredictable and fluctuating environments.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.NT-K and BR were supported by NSF-BSF Grant: BSF-2019647, NSF-1951826. NT-K and WEB were supported by NSF-2013692. LDM: was supported by NSF OCE- 1928771 and NSF OCE-2109622. BR would like to thank Alex and Ann Lauterbach for funding the Comparative and Evolutionary Immunology Laboratory. The work of BR was supported by Israel Science Foundation (ISF) numbers: 1416/19 and 2841/19. BR has received funding from European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program under grant agreement No. 948476. BR has received funding from a HFSP grant (RGY0085/2019).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. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


With the advancement of sequencing technologies, there has been a rapid accumulation of data making it difficult to decipher the key genetic elements, their nature, and alterations for progression or regression of cancers. Further, enlightening an optimum gene set for optimal classification, diagnosis, and prognosis of cancer types is difficult without reliable and efficient computational tools. Progress in bioinformatics tools is parallel with sequencing data accumulation. Computational tools are being continuously refined and developed to meet specific challenges in cancer biology, the primary problem with them is that their accuracy is often insufficient for clinical use , breast cancer, esophageal cancer (EC), in colorectal cancer (CRC), metastatic melanoma, and multiple myeloma. The computational pipelines developed in these studies have a great potential to be extended to uncover novel therapeutic targets of other cancer types.Chen et al. proposed to use machine learning algorithms to identify primary lesions for primary metastatic tumors. The fundamental idea behind their model is that different tumor types exhibit specific expression profiles for certain genes, which could be captured through machine learning models to classify the primary lesions. In essence, they used gene expression data from TCGA and GEO, analyzed and processed it to obtain a relatively suitable machine learning model followed by evaluation of the efficiency of diagnosis of primary lesions. They used XGBoost for classification and their results revealed that by combining tumor data with machine learning methods, the classification of different cancers can be achieved with specific accuracy, which can be used to predict the location of primary metastatic tumors. Cui et al. developed a novel pipeline, which not only compares two single-cell clusters but also calls for differential gene expression, coexpression network modules, etc. They used two single-cell data sets; Usoskin from the GEO database and Xin dataset of the human pancreas. Different types of analysis were then performed sequentially through a variety of computational tools to create a smooth pipeline. The pipeline implements DEsingle and SigEMD for differential gene expression analysis, DGCA for differential correlation analysis, WGCNA for network analysis, and DNA for differential network analysis. This pipeline is very effective to unravel the key differences between cell clusters and cell types and provides one place for easy computational analysis of single-cell data sets. Zhao et al. designed an Autoencoder-based computational framework, which could capture both intrinsic and extrinsic features of melanoma. They used the expression data of the TCGA metastatic melanoma gene RNA-seq dataset from Firehose and decomposed it into a small number of representative nodes. Further, microarray datasets from GEO for melanoma were used for prognosis analysis. They identified many nodes that were significantly associated with the prognosis of melanoma patients using Cox proportional hazard models. A tumor-intrinsic (TI) signature and a tumor-extrinsic (TE) signature were established from the two most prognostic nodes. Both these signatures highly predicted the patient's overall survival. In addition, the TE signature successfully predicted the response of patients to immunotherapy techniques. Using an integrative approach of somatic mutations and gene expression data, Jiang and Jin proposed a novel method for the identification of breast cancer-associated mutated genes. The fundamental theme behind their analysis is to first create a mutation matrix data and evaluating mutation frequency for each gene, then to create a gene expression matrix with expression values for each gene. Finally, both data sets are mapped to identify the co-expression profile. Their results indicated that this integrative approach is effective in breast cancer classification.Four studies focused on the usage of specific computational approaches to address diverse problems. Yuan et al. aimed to create a new molecular classification and to provide new therapeutic targets for GBM. They performed an integrated analysis based on the SNPs, DNA copy, DNA methylation, and mRNA expression profile data of 117 patients. The data was obtained from the TCGA database and Genomic Data Commons database (GDC). MutSigCV and GISTIC modules from GenePattern were used in the analysis of driver genes and landmark CNV events in GBM, respectively. Using the cluster of cluster analysis (CoCA), they found two novel subtypes, HX-1 and HX-2 depicting three variable methylation positions and fifteen gene mutations. These subtypes may act as potential prognostic biomarkers for patients with glioblastoma. Zhang et al. tried to classify glioblastoma subtypes on the basis of different degrees of gene methylation. They used the methylation datasets from Gene Expression Omnibus (GEO), identified the methylation loci, which served as potential biomarkers to classify and annotate the different GBM subgroups. They used powerful machine learning algorithms to achieve their goals. Monte Carlo feature selection (MCFS) and incremental feature selection (IFS) methods were used to extract 4,100 essential methylation sites and support vector machine (SVM), random forest (RF) metaclassifier, and repeated incremental pruning to produce error reduction (RIPPER) were used during classification. Functional enrichment analysis of these dysmethylated genes using GO and KEGG databases revealed several biological functions related to GBM classification.Two studies focused on the classification and prognosis of glioblastoma multiform (GBM), which lacks accurate prognostic markers and drug targets. Huang et al. aimed to evaluate the significance of genetic alteration (CDK4 amplification) in the prognosis of esophageal squamous cell cancer (ESCC). Through tissue microarray and fluorescence in situ hybridization they found that among the investigated 520 patients with ESCC, 8.5% exhibiting CDK4 amplification showed a negative correlation with disease progression and significantly better survival. Thus, they declared CDK4 amplification as an independent prognostic factor for the survival of patients with ESCC. With the aim of identifying the novel DNA damage and repair-related prognostic genes in colorectal cancer (CRC), Wang et al. identified 1,545 genes related to DNA damage and repair. They used gene expression data of 471 COAD (Colon adenocarcinoma) and 41 normal samples from The Cancer Genome Atlas-Colon adenocarcinoma (TCGA-COAD) and 4 datasets of colon cancer from the GEO database. Following the gene set enrichment analysis (GSEA), the prognostic relevance of the individual genes was evaluated through Cox regression analysis on the TCGA-COAD dataset. A set of 12 genes related to DNA damage-and-repair were identified, which classified COAD patients into high and low-risk groups. Genes co-expressing with these 12 genes were identified through Pearson's correlation method. WGCNA with Topological Overlap Matrix (TOM) was used to construct the gene-coexpression network. Functional annotation of the functional gene modules was carried out using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The gene set identified in this study has great potential in the prognosis, and treatment of CRC. Further, using The Cancer Genome Atlas (TCGA)-Multiple Myeloma Research Foundation (MMRF) dataset as a training dataset, Wang et al. analyzed the expression profiles through R package limma and evaluated the prognostic relevance of each gene through univariate Cox regression. Risk predictions were established through Lasso and stepwise Cox regressions followed by validation using GEO datasets. A set of eight RBP hub genes were identified, which classified multiple myeloma patients into high- and low-score groups. Functional analysis through Gene Ontology, KEGG Enrichment Analysis, and Gene Set Enrichment Analysis (GSEA) revealed that the major pathway through which RBP's could lead the development of myeloma may be the spliceosome pathway.Three studies aimed to reveal the novel prognosis-related signatures in different cancers. Esophageal cancer (EC) is a global fatal disease with a poor prognosis. Yang et al. used the whole exon sequencing and immune checkpoint analysis of five patients with ciliated muconodular papillary tumors (CMPT) to elucidate the molecular details and histogenesis in CMPT. They observed 77 gene mutations in the patient's tumor tissue and 31 mutations in the border tissue. Interestingly, CMPT shared the same phylogeny with cancer tissue. These results suggest the CMPT indeed are neoplastic processes with immune escape and have malignant potential. Recent studies have revealed that the clinical outcome of multiple cancers could be predicted through tumor mutational burden (TMB). To ascertain the relationship between TMB level and clinical features and outcomes of lung Adenosquamous carcinoma (ASC), Cheng et al. used NGS and immunohistochemistry approach and identified 95 unique genes with somatic variations from a total of 475 genes evaluated. TMB was found to be associated with pathological stages, invasion of lymph node, and overall survival but not with age, sex, smoking history, and tumor size in lung ASC. Moreover, no correlation between TMB and mutations in TP53 and EGFR was observed. This study, therefore, provided an evidence that higher TMP correspond to lesser survival and higher lymph node invasion.Two studies identified mutations in driving cancers. Su et al. developed a new version of U-Net called multiscale U-Net (MSU-Net), which employed a new image segmentation architecture. It is based on Multi-scale blocks composed of convolution sequences with different receptive fields, which facilitates extraction of more information with diversified features. Their results showed that MSU-Net enabled significant improvement of semantic segmentation. MSU-Net integrates multiple convolution sequences having receptive fields of different sizes, which produces more conspicuous object features during forward propagation. Besides, MSU-Net is flexible enough to be integrated with other network structures. MSU-Net showed improved results, with 5-fold cross validation when applied on five biomedical image segmentation datasets; (1) 30 serial section Transmission Electron Microscopy (ssTEM) images (512 × 512 pixels) of the first instar larva ventral nerve cord (VNC) of the Drosophila, (2) The Breast Ultrasound Dataset B (BUL) comprising of 163 ultrasound images (760 × 570 pixels) of breast lesions, (3) 800 Chest X-ray (CXR) images  from the standard digital image database for Tuberculosis, (4) 2,594 RGB images of skin lesions , and (5) Nuclei Segmentation (NS) dataset from The Cancer Genome Atlas (TCGA) comprising of 30 digitized Hematoxylin and Eosin-stained frozen sections (512 × 512 pixels). This imaging technology may perform better in tracing, diagnosis, prognosis, and possible treatment of different cancer types.One study focused its interest on improving the existing medical imaging technology, which is a commonly useful approach in disease diagnosis and progression. With rapid advancements in deep learning, medical imaging technology has been revolutionized. Most medical imaging techniques involve encoder-decoder system, the classical architecture of which is implemented in U-Net. Several modified versions of U-Net have been introduced till now, all of which have two major limitations; loss of diversity features caused by fixed receptive field of the convolution kernel and loss of information when a single convolutional sequence is used in extracting features at each scale. With the aim of overcoming these limitations, This editorial was designed by LK and written by LK and AM. WM and JC revised the editorial. All authors made a direct and intellectual contribution to this topic and approved the article for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Gene expression and translation have been extensively studied in human post-mortem brain tissue from subjects with psychiatric disease. Post-translational modifications (PTMs) have received less attention despite their implication by unbiased genetic studies and importance in regulating neuronal and circuit function. Here we review the rationale for studying PTMs in psychiatric disease, recent findings in human post-mortem tissue, the required controls for these types of studies, and highlight the emerging mass spectrometry approaches transforming this research direction. Psychiatric disease imparts a substantial burden on the global population. For example, depression , schizopWhile many areas of research benefit greatly from the use of animal models, polygenetic psychiatric disorders are difficult, if not impossible to model in animals, complicating the investigation of disease etiology. In an effort to elucidate the molecular mechanisms driving these structural and functional impairments the field has turned to transcriptomic and proteomic analyses of human post-mortem brain tissue to quantify disease associated differences in transcripts  and protMore recently, multi-omics analyses, grounded in GWAS, have identified quantitative trait loci (QTLs) for common risk variants associated with gene expression (eQTLs)  and protPost-translational modifications have been studied in neurological disorders, most notably Tau hyperphosphorylation in Tauopathies such as Alzheimer’s disease , but theUnbiased genetic analyses firmly implicate PTMs in the etiology of a number of psychiatric diseases. The most recent report from Investigations of transcript levels in human post-mortem brain tissue from autism spectrum disorder and schizophrenia subjects further implicate PTMs. The most recent meta-analysis of RNAseq studies from PsychENCODE  found thThese findings are not surprising as schizophrenia and autism spectrum disorder are both widely viewed as developmental synaptopathies  and a muBy the early 2000s dendritic spines  and NMDAFinally, The studies above measured static phosphorylations, presumably preserved at death. But a few adventurous groups have pushed these studies further, attempting to capture dynamic phosphorylation activities in human post-mortem brain tissue. In two publications, Hahn and Wang combined targeted phosphorylation studies with a post-mortem tissue-stimulation paradigm to identify phosphorylation differences in schizophrenia after receptor stimulation . In the A key driver underlying the incomplete correlation between mRNA and protein abundance is the fact that protein turnover is dynamic. The principal mechanism of turnover is the ubiquitin-proteasome system in which polyubiquitinated proteins are targeted to the proteasome for degradation. Additionally, the ubiquitin-proteasome system regulates synaptic protein stability and is essential for LTP and learning . Thus, wWhile this review has focused on ubiquitination and phosphorylation a growing body of work implicates additional PTMs such as glycosylation and myristylation in schizophrenia, reviewed in detail in The studies reviewed above suggest several points: (1) That differences in PTMs can be observed in brain tissue from subjects with psychiatric disease; (2) that levels of multiple classes of PTMs are altered across multiple protein families; and (3) when tested in forward genetic models, individual PTMs can significantly impact disease relevant biology such as glutamatergic signaling and dendritic spine plasticity, that could not be predicted by genetics, transcriptomics, or even protein quantification. For example, MAP2 is not found at any schizophrenia risk loci and its protein levels are unaltered in schizophrenia tissue, yet it is hyperphosphorylated at multiple sites in schizophrenia and modeling just one of these sites induces a loss of dendritic spines.via mostly targeted approaches highlights the need for broad and systematic investigations of PTMs in psychiatric disease. Specifically, next generation studies should be rigorously designed to catalog post-mortem effects on individual PTM sites, be performed in well powered and well-balanced cohorts, utilize state-of-the-art mass spectrometry approaches, target selected brain areas, cortical layers, cell types, and microdomains, and take advantage of new informatic and statistical approaches to multi-omic integration to map associations between genes, multiple PTMs, and phenotypes.The breadth of the PTM alterations observed in schizophrenia The impact of post-mortem interval  on molecular integrity has long been appreciated and three main strategies have been employed to account and control for this confound. (1) The effect of PMI on individual PTM sites can be modeled, using either mouse  or humanAdvances in mass spectrometry instrumentation and sample preparation techniques continue to increase the throughput, breadth and depth of PTM coverage, and some of these approaches have begun to see use in human post-mortem brain tissue. Next generation proteomics instruments such as the timsTOF (Bruker) and Orbitrap instruments (ThermoFisher) with increased scan speeds facilitate deep coverage of modified peptides. For example, Protocols to enrich phosphopeptides and glycopeptides from tryptic digests are now well developed and can be accomplished with high efficiency using commercially available kits and specialized liquid handling robots with pre-programmed proteomic applications, such as the AssayMAP BRAVO (Agilent), enabling both throughput and quantitative depth. Quantification of protein ubiquitination initially proved more challenging in standard proteomic work flows as trypsin digests off the larger ubiquitin side chain leaving only a lysine bound gly-gly which was difficult to capture. However, the recent availability of commercial antibodies and refined sample preparation now allow for deep profiling of protein ubiquitination in human samples as well; for example, over 14,000 ubiquitination sites were recently quantified in human tumor cells .As reviewed above, the activity, structure, and molecular pathologies associated with psychiatric illness are highly spatially localized, displaying brain area, layer, cellular, and microdomain specificity. Multiple groups have used mass spectrometry to quantify protein levels in cortical layer captures  and synaKeeping a pace with the advancements in instrumentation, the last few years has seen the release of multiple software packages for exploring PTMs in the context of known kinase-substrate relationships, kinase motifs, protein–protein interactions, and protein networks. Packages such as iGPS  and KEA2In closing, PTMs are implicated in the etiology of neuropsychiatric diseases by unbiased genetic and transcriptomic studies, most notably autism spectrum disorder and schizophrenia. When investigated in human post-mortem brain tissue using targeted approaches, robust alterations in phosphorylation, glycosylation, and ubiquitination are observed in psychiatric disease, suggesting a much broader set of changes, with likely associations between different PTMs as well as the genome, transcriptome, and proteome. Advances in mass spectrometry instrumentation and proteomic sample preparation methods now allow for sufficiently powered studies to map these interactions, which, when combined with emerging informatics tools, will surely provide insight into the etiology of many psychiatric diseases as PTMs are the ultimate mediators of so many neuronal and circuit activities.MM wrote the first draft of the mini review. MG, RS, and MM revised and reviewed the second draft of the mini review. All authors contributed to the article and approved the submitted version.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.


Nowadays patients affected by locally advanced breast cancer and particular subtypes of early breast cancer may benefit from neoadjuvant chemotherapy (NAC) before surgery with different advantageous, like reduction of tumor size and prognosis improvement. Pathological complete response to NAC is very variable amongst all different histological and immunophenotypic subtypes of breast cancer and its correct assessment by imaging is crucial for treatment planning, as patients could be addressed to conservative or demolitive breast surgery with reconstruction. Advanced imaging techniques, such as MRI and nuclear medicine, recently contributed to the prediction of chemotherapy response in the early phase of NAC, to avoid side effects and psychological implications of the oncological treatment in patients who are supposed to be unresponsive. This review article aims to compare different imaging techniques for both assessment and prediction of response to NAC and explain the new revolutionary contribute offered by Artifical Intelligence in this field. Neoadjuvant chemotherapy (NAC) is becoming the standard of care for locally advanced breast cancer, aiming to reduce tumor size before surgery. Unfortunately, less than 30% of patients generally achieve a pathological complete response and approximately 5% of patients show disease progression while receiving NAC. Accurate assessment of the response to NAC is crucial for subsequent surgical planning. Furthermore, early prediction of tumor response could avoid patients being overtreated with useless chemotherapy sections, which are not free from side effects and psychological implications. In this review, we first analyze and compare the accuracy of conventional and advanced imaging techniques as well as discuss the application of artificial intelligence tools in the assessment of tumor response after NAC. Thereafter, the role of advanced imaging techniques, such as MRI, nuclear medicine, and new hybrid PET/MRI imaging in the prediction of the response to NAC is described in the second part of the review. Finally, future perspectives in NAC response prediction, represented by AI applications, are discussed. Neoadjuvant chemotherapy (NAC) is becoming the standard of care for locally advanced breast cancer  aiming tThus, early prediction of the response to NAC could be an important weapon for individualized medicine. A prediction model could avoid patients to be overtreated with useless chemotherapy cycles which are not free from side effects and psychological implications. It is likewise crucial to accurately assess the response to treatment after NAC in order to establish the most appropriate surgical approach.Imaging plays a fundamental role in the evaluation of patient candidates to NAC for the evaluation of primary tumor and axillary lymph-node status. Indeed, the main aim of imaging, particularly by means of ultrasound (US), digital mammography (DM)/digital breast tomosynthesis (DBT), is the assessment of the response to treatment and the estimate of residual tumor, both influencing patients’ prognosis and surgical strategy . The intThe aim of this review is to elucidate and compare the role of conventional and advanced imaging techniques in assessing and predicting the response to NAC as well as to illustrate future perspectives represented by AI applications. A comprehensive summary of the major imaging techniques employed for the assessment and prediction of response to treatment is reported in Patients who undergo NAC are essentially monitored by a clinical breast examination (CBE), mammography (DM), ultrasound (US), and/or magnetic resonance imaging (MRI). The accurate assessment of breast tumor and regional lymph node response to preoperative systemic therapy is crucial and the selection of imaging methods prior to surgery should be determined by the multidisciplinary team, according the recent NCCN guidelines . All theThe accuracy of DM-based post-treatment evaluation depends upon specific tumor morphological characteristics. In particular, it is higher for lesions who appear well-circumscribed on the pre-treatment examination. The most reliable indicators of treatment response are decrease in size and density, whereas calcifications and spicules are the biggest challenge for imaging interpretation. Indeed, several authors correlated mammographic microcalcification with surgical specimen and concluded that residual microcalcifications on DM could be due to both residual tumor   and treaKim et al.  demonstrDBT has proven to be superior to DM for the evaluation of tumors size, especially in case of small lesions and dense breast . MoreoveUS is considered a more accurate method than CE or DM in assessing tumor size and in the monitoring of residual breast tumors, but operator dependency and shortage of qualified personnel have been an issue in hand-held US . ConflicIn a recent retrospective study by Makanjuola et al. , US has New technologies have been introduced in breast US imaging such as the automated breast ultrasound (ABUS) which provides 3D images using wider probes. Shin et al.  enhancedNAC monitoring methods such as DM or US or physical examination are not free from limitations . SometimFor instance, Taxanes determine a decrease of enhancement of the tumor and the whole breast tissue as it has a specific anti-angiogenetic effect . Functio37), while DWI-MRI allows the calculation of apparent diffusion coefficients (ADC)—a quantitative measure of the diffusivity of water—providing information related to tumor cellularity and the integrity of cell membranes, being sensitive to intra-tumoral changes induced by chemotherapy. MRI has been proved to accurately evaluate residual tumor after NAC with high sensitivity (76–92%), specificity (60–89%), and accuracy (76–90%) . . 61]. Although heterogeneous results regarding the use of DCE-MRI parameters are reported, it seems that the combination of quantitative perfusion parameters such as Ktrans, Kep, and Ve reflecting tumor permeability and cell density can early predict pCR during NAC  62]. . 62]. In this setting, Tudorica et al.  confirme18F-FDG PET/CT is routinely used for staging, recurrence evaluation, and treatment response. Whole-body 18F-FDG PET/CT enables metabolic activity assessment of breast tumors. Standardized uptake value (SUV) seems to correlate with histology as it is higher in high-grade invasive ductal carcinomas, triple negative, and erb-2 negative tumors and lower in low-grade lobular carcinoma. Furthermore, SUV is significatively lower in patients who experience pCR after the second cycle of chemotherapy [18F-FDG PET/CT. In their study including 67 patients, they found that volume-based metabolic variables obtained with 18F-FDG PET/CT such as metabolic tumor volume (MTV) and total lesion glycolysis (TLG), unlike SUV based variables , were good predictors of both NAC response and prognosis in locally advanced breast cancer. The main limitation of PET is the assessment of small tumors as it has low spatial resolution. PEM could overcome such limit as it is accurate for the evaluation of small lesions. In particular, Soldevilla et al. used PEM for interim prediction of the response to NAC in a retrospective study enrolling 108 patients. Though further studies are required to validate these results, SUVmax and lesion to background (LTB) showed strong correlation with pCR [Hybrid-Imaging using PET/CT could be extremely useful in early response prediction as it assesses both morphological and functional cancer features. In particular, tumor cells change their metabolic expression after NAC, thus the assessment of glucose uptake rates might be of clinical value. Indeed, otherapy . Garcia otherapy  also anawith pCR . 18F-FDG PET/MRI could be used to predict non-pCR after the first cycle of NAC. Specifically, the sensitivity significantly improved with the addition of MRI to PET parameters [PET/MRI is the hybrid newest imaging technique which allows the simultaneous collection of morphologic, metabolic, and functional parameters with higher contrast resolution compared to PET/CT. It could be used for staging locally-advanced breast cancers and for monitoring and evaluating response to neoadjuvant and systemic chemotherapy . A recenrameters . In a rerameters , 14 womerameters  demonstrrameters  also sho18F-FDG-PET [Different imaging methods have been explored in literature to detect early response to NAC using AI, including QUS, diffuse optical spectroscopy (DOS) , 18F-FDG-FDG-PET , but a g-FDG-PET ,75.Differently from MRI and PET, which may have limitations in repeated use due to high costs and contrast agent/tracer injection, US is relatively inexpensive and safe for patient management. In particular, newest advanced QUS methods could be able to early depict changes in tumor microstructure without contrast agent administration. Fernandes et al.  used a mp < 0.002) using pretreatment MRI performed in 288 patients. Braman et al. retrospectively evaluated 117 patients who underwent DCE-MRI and subsequent NAC. Combined intratumoral and peritumoral texture analysis-based radiomic approach was proved to predict pCR to NAC, independently of prior knowledge of receptor status suggesting their validity as response predictor [Several studies analyzed the potential of AI with multiparametric MRI to predict response to early predict NAC. In this light, 38 patients underwent multiparametric MRI before and after two cycles of NAC in a study by Tahmassebi et al. with the extraction of both qualitative and quantitative parameters . In detaredictor . Moreoveredictor . Chamminredictor . Accordiredictor . 18F-FDG PET offers the potential to non-invasively characterize tumor heterogeneity, a factor strongly related to tumor growth and resistance to medical treatments. However, reliable biomarkers have not been established yet. In this context, Li et al. recently published a retrospective study including 100 breast cancer patients who received NAC [18F-FDG PET/CT scans were able to predict pCR after NAC especially when combined with patient age or Ki 67 levels from pre-treatment core needle biopsy specimens, as also demonstrated in the last prospective study by Luo et al. [Radiomics applied to ived NAC . Radiomio et al. . In theio et al.  supporteAssessment of residual tumor and prediction of response in patients with locally advanced breast cancer undergoing NAC are clinically relevant to obtain the best outcome. Advanced imaging techniques are making headway in the assessment of such patients along with morphological and functional modalities. The possibility to extract information reflecting tumor biology from medical images could aid in the early identification of patients who will benefit from NAC, optimizing the response to treatment and avoiding unnecessary toxicity. In this perspective, hybrid imaging modalities and AI may have several and attractive future applications.