Adverse pregnancy outcomes are considered significant health risks for pregnant women and their offspring during pregnancy and throughout their lifespan. These outcomes lead to a perturbated in-utero environment that impacts critical phases of the fetus’s life and correlates to an increased risk of chronic pathological conditions, such as diabetes, obesity, and cardiovascular diseases, in both the mother’s and adult offspring’s life. The dietary intake of naturally occurring antioxidants promotes health benefits and disease prevention. In this regard, maternal dietary intake of polyphenolic antioxidants is linked to a reduced risk of maternal obesity and cardio-metabolic disorders, positively affecting both the fetus and offspring. In this work, we will gather and critically appraise the current literature highlighting the effect/s of the naturally occurring polyphenol antioxidant resveratrol on oxidative stress, inflammation, and other molecular and physiological phenomena associated with pregnancy and pregnancy conditions, such as gestational diabetes, preeclampsia, and preterm labor. The resveratrol impact on prenatal complications and pregnancy-associated structures, such as the fetus and placenta, will also be discussed. Finally, we will draw conclusions from the current knowledge and provide future perspectives on potentially exploiting resveratrol as a therapeutic tool in pregnancy-associated conditions. Adverse pregnancy outcomes are considered major health risks for pregnant women and their offspring during pregnancy and throughout their lifespan, with several disorders recognized, including gestational diabetes, preterm birth, fetal-growth restriction, and hypertensive disorders of pregnancy such as gestational hypertension, pre-eclampsia, and related conditions ,2. TheseDietary intake of naturally occurring antioxidants has been linked to disease prevention and health benefits ,5,6,7. IThis literature review will gather and discuss the current experimental evidence highlighting the effect/s of resveratrol on inflammation, oxidative stress, and other molecular and physiological phenomena associated with pregnancy and pregnancy-associated complications, including preeclampsia, gestational diabetes, and preterm labor. The effect of resveratrol on prenatal issues and pregnancy-associated structures such as the placenta and fetus will also be discussed. Finally, conclusions on the current knowledge and future directions on potentially exploiting resveratrol as a therapeutic tool in pregnancy-associated conditions are provided to the readers.Due to its numerous pharmacological activities, resveratrol’s effects on humans are widely studied. Nonetheless, results from the available human clinical trials are so far contradictory concerning the protective effects of resveratrol against diseases and their sequelae . SimilarIn human clinical trials, resveratrol was proven to be generally tolerated, yet some adverse effects, including nephrotoxicity and gastrointestinal problems, were reported ,35. For Both in vitro and in vivo resveratrol’s biological effects are associated with its hermetic behavior, where low resveratrol doses induce beneficial effects while higher doses usually have a harmful effect . EvidencInterestingly, maternal resveratrol intake has been reported to have potential beneficial effects in adverse human pregnancies  and has A systematic data analysis across different species and dosages indicated that resveratrol consumption could decrease inflammation and oxidative stress in placental and embryonic tissues . OxidatiIn this regard, studies in murine models have shown that oxidative stress biomarkers and metabolic dysfunction caused by a low-protein diet in the mother, placenta and offspring could be improved by a resveratrol-rich diet in pregnant individuals . Anti-atEpigenetic regulation represents another potential mechanism related to the maternal resveratrol intake effects and modulates methylation and acetylation processes affect gene expression . Among tResveratrol is also recognized as a Sirtuin 1 (SIRT1) activator. The Sirtuin proteins family has a beneficial impact on longevity mainly linked to their effects on metabolic control since they are substantially contributing to lipid and glucose regulation via the deacetylation of crucial metabolic signals relevant to the activated.The Sirtuin proteins family has a beneficial impact on longevity primarily related to their effects on metabolic control as they substantially contribute to lipid and glucose regulation through the deacetylation of crucial metabolic signals related to the activated protein kinase-SIRT1-PPARG coactivator-1α axis ,76. In tPregnancy is known to cause increased levels of oxidative stress, as an aspect of the systemic inflammatory response, resulting in high-circulated amounts of ROS, with the placenta representing the main source . PE is aPE-associate oxidative stress originates from the placental impairment caused by hypoxia-reoxygenation imbalance, lower efficiency of both free radical scavengers and antioxidant enzymes, impaired angiogenesis, decreased nitric oxide (NO) bioavailability vascular endothelial dysfunction, cardiovascular complications, and exaggerated inflammatory response ,87,88,89PE etiology is not fully understood, however, this condition is believed to be associated with impaired uterine artery blood flow . In thisIt has been recently shown that circulating SIRT1 is reduced during PE . In thisSeveral studies suggested that the resveratrol-associated anti-hypertensive effect is mainly related to its capacity to attenuate blood pressure and hypertension symptoms. Interestingly, a clinical trial with 400 PE patients investigated the outcome of a nifedipine/resveratrol combined treatment against PE, revealing that resveratrol supplementation significantly reduced the time needed to control blood pressure with a better result in the group treated with both nifedipine  and resveratrol, which also had a significantly delayed time between the hypertensive crises intercourse  . In contConsistent with this, other studies indicated that resveratrol anti-hypertensive properties are also related to its ability to inhibit the release of endothelial dysfunction-associated anti-angiogenic factors and that their elevation is a main player in placental oxidative stress and inflammation during the maternal endothelial dysfunction in PE . For insGDM is a type of diabetes characterized by an impaired glucose tolerance that occurs during pregnancy . AlthougStudies also demonstrated that GDM is often related to adverse metabolic health outcomes in offspring as a common pregnancy complication. In a GDM model of female Sprague Dawley rats fed with a high-fat and sucrose diet, Brawerman et al.  showed tIt is well known that insulin resistance (IR) caused by insufficient insulin secretion during pregnancy, may lead to metabolic disorders, mainly GDM and related pathologies . AttentiMaternal obesity is also a metabolic complication that rates high maternal and fetal oxidative stress and inflammation. Hypothetically, offspring of obese mothers are at greater risk of developing obesity, diabetes, and cardiovascular disorders in adult life , with riA recent study conducted in obesity murine models revealed that maternal resveratrol supplementation (20 mg/kg/day) improves maternal metabolism and reduces the placental and liver oxidative stress of mothers and fetuses in a sex-dependent manner . CompareOn the other hand, diabetes-associated lipid profile disruption may indirectly affect embryogenesis and organogenesis , and theOverall, the above-mentioned studies evaluated the short-term effects of maternal resveratrol intake in diabetic and related-metabolic disorders experimental models Several studies supported resveratrol’s protective and therapeutic effects at the prenatal stage. In fact, offspring may show increased susceptibility to certain diseases due to prenatal or intrauterine conditions, which may be efficiently reversed by resveratrol. In this section, we present several examples of disease susceptibility due to intrauterine modifications and the potential role of resveratrol in reversing this susceptibility. These include susceptibility to metabolic, cardiovascular, and neurological disorders.Non-alcoholic fatty liver disease (NAFLD) is a hepatic metabolic disorder  associatOffspring susceptibility to hypertension may originate at the prenatal stage due to maternal conditions. A study showed that the HF diet exacerbated maternal L-NAME treatment-induced programmed hypertension of rat offspring . This wa−1; Research Diet) improved their metabolic function prenatally challenged by hypoxia and postnatally by HF diet [It is accepted that prenatal hypoxia increases offspring susceptibility to cardiovascular disease and metabolic disorders ,152 such HF diet . Molecul HF diet .Prenatal stress may predispose neurological complications in offspring. In a study realized on rat offspring, prenatal gestational stress increased lipid peroxidation and protein oxidation and decreased antioxidant activity and NO levels . In addiAutism spectrum disorder (ASD) is a neurological disorder characterized by decreased social communication and interaction . While lThe placenta is a large organ that develops in the uterus during pregnancy. It provides oxygen and nutrients to the fetus and removes waste products from its blood. It plays a crucial role in fetal organ development and fetal growth . In addiNoteworthy resveratrol administration during pregnancy ameliorates placental oxidative status, which is achieved through a plethora of biological effects ,175,176.The placenta favors the flow toward the fetus of epigenetic alterations induced by the mother’s conditions, such as GDM and obesity ,179. TheStudies on non-human primates have also shown that resveratrol reduced placental inflammation and improved glucose metabolism ,144. A sOverall, the presented data indicate that dietary resveratrol supplementation during pregnancy improves the placental status, which is beneficial for reproductive performance and the offspring’s well-being. However, long-term clinical trials on pregnant women are warranted to reinforce resveratrol’s beneficial effects on the placenta and placenta-associated functions.Preterm birth (PTB) (<37 weeks of gestation) is considered a leading cause of newborn death and a risk factor for short and long-term adverse health outcomes. Although the etiology of PTB remains unclear, it is thought that an inappropriate increase in net inflammatory load seems to be a pivotal key . Genitalp < 0.05) in the uterus of LPS-treated mice and parallelly reduced the expression of LPS-induced pro-inflammatory agents, such as iNOS (p < 0.05), COX-2 (p < 0.05), prostaglandin E2 (PGE2) (p < 0.05), and anandamide (AEA) (p < 0.05). Interestingly, resveratrol administration also prompted ameliorative changes in the LPS-altered uterine endocannabinoid profile [Despite the immensity of research into PTB pathophysiology, the resveratrol administration effect on this disorder remains unclear, although few studies suggested that resveratrol decreases PTB rate mainly via the suppression of inflammation and infection complications. Regarding the studies included in this review, only a few investigated the correlation between resveratrol consumption and PTB. In a study realized by Bariani et al. , in vivo profile . In addi profile ,193,194, profile , and mat profile , which t profile , leading profile , resvera profile . Moreove profile .It is well established that SIRT1 is mainly localized in the nucleus to exert its antiapoptotic effects during oxidative stress or toxic substance-induced damage, and its amount increases in the nucleus to protect the cell from apoptosis. In a tentative to identify the protective role of SIRT1 in PTB and the role of resveratrol to ameliorate its dysregulation, a study realized on PBMCs cells sampled from an established hyperoxia model of premature infants administered with different amounts of oxygen at birth, demonstrated a concentration-dependent increase in SIRT1 translocation rates, ROS and MDA levels. Interestingly, resveratrol (6 µmol/L) treatment managed to attenuate hyperoxia-associated outcomes resulting in opposite effects .Overall, the effects of resveratrol in PTB complications appear to be based on its capacity to counteract infections, inflammation, and oxidative stress-related outcomes. Nevertheless, further investigations including clinical trials are needed to better elucidate the molecular underlying mechanisms of action related to the beneficial effect of resveratrol intake on PTB.The “fetal basis of adult disease” hypothesis is now considered a keystone in the development of important diseases in adult/advanced age, such as cardiovascular and metabolic diseases. These events are associated with epigenetic dysregulations, and they are mainly supported by alterations in the fetus development during pregnancy and caused by non-optimal conditions in the uterus, such as GDM, maternal malnutrition, and functional placental deficit ,201.Resveratrol pregnancy intake appears to be able to reverse the negative effects on the fetus caused by in-utero adverse conditions and induce a positive impact on adult life health. Although studies have been carried out on different species and with different dosages and routes of administration, the overall data indicate that resveratrol is capable of providing positive effects on fetal development in different gestational phases .Prenatal exposure to environmental stress can also lead to greater susceptibility to clinical disorders in adulthood. This is the case of fetal exposure in utero to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leading to alterations in the differentiation of T cells in the thymus and to a great susceptibility to autoimmune diseases . TCDD caMothers exposure to ethanol during pregnancy may also induce various disorders in their offspring, known as fetal alcohol spectrum disorders (FASD), which mainly affect the cerebellum from developing. A study by Kumar et al.  demonstrSeveral benefits of resveratrol administration  during pregnancy and fetal development were notable in the study by Darby et al. , where tAnother positive effect of resveratrol on fetal outcomes is its ability to increase fetal hemoglobin levels (HbF) . Among tMoreover, a recent work by Bosquesi et al.  reportedResveratrol owns multiple positive health effects on different metabolic parameters, but it also possesses many different negative activities on the cellular life of various tissues/organs in different animal species ,214,215.Resveratrol has been reported to possess numerous benefits against complicated pregnancies, with its activity elicited by regulating different physiological and cellular functions and modulating multiple intracellular signaling pathways implicated in pregnancy and pregnancy-associated conditions. Resveratrol demonstrated potent antioxidant activities by blocking DNA damage, regulating antioxidant enzymes, and modulating redox-regulated intracellular signals. Resveratrol also showed anti-inflammatory properties by inhibiting pro-inflammatory signaling pathways and the secretions of pro-inflammatory cytokines and growth factors. Resveratrol has the capability to cross the placenta, improve its oxidative status and directly affect the fetus during the gestational period producing beneficial effects for the reproductive performance and the offspring’s well-being. Resveratrol may offer potential therapeutic benefits for PE due to its antioxidative stress/anti-inflammatory properties that prevent cells from undergoing apoptosis and mediates a protective effect by decreasing blood pressure and attenuating vascular endothelial injury. Interestingly, resveratrol mediates a positive response in blood glucose regulation and attenuates GDM-related complications.Despite the multiple reported benefits, the use of resveratrol in pregnancy needs to be better investigated to dissolve the high-dose-related toxicity issues highlighted by some papers and the intricate information derived from the available literature analysis. Indeed, although clinical trials indicate resveratrol safety for human consumption, results from the same studies are inconclusive regarding resveratrol’s protective effects against diseases and their complications. In particular, due to the vast difference in experimental models employed, duration of treatments, dosages, routes of administration, and formulations, the final interpretation of resveratrol effects on pregnancy and associated complications remains to be extrapolated.Particular attention should be directed to performing systematic long-term clinical trials on pregnant women using determined resveratrol dosages to possibly reinforce and ultimately demonstrate resveratrol’s protective and therapeutic role. Specifically, the optimal resveratrol dosages to be employed in humans remains to be clarified. Indeed, ranges of biologically active resveratrol concentrations in vitro and in vivo experimental models differ enormously; hence, based on the currently available data, it is difficult to extrapolate the resveratrol concentration/s that provide the maximum beneficial effect in human subjects without provoking toxicity. This aspect is even more critical considering the resveratrol hormetic properties that tightly associate its final biological outcomes to the employed concentrations. Not to mention the interaction of resveratrol with the cellular and body redox state, which also influences its final effect in being beneficial or harmful. Furthermore, due to its poor bioavailability, as performed for other natural compounds ,218,219,Overall, the data presented in our review indicates that resveratrol pregnancy consumption has numerous benefits on metabolic health for pregnant women and their offspring therapeutic approach in pregnancy-related pathological conditions such as elevated inflammation, preeclampsia, and GDM . Therefo


However, nearly all such whole-embryo atlases of embryogenesis remain limited by sampling density—i.e., the number of discrete time points at which individual embryos are harvested and cells or nuclei are collected. Given the rapidity with which molecular and cellular programs unfold, this limits the resolution at which regulatory transitions can be characterized. For example, in the mouse, there are typically Drosophila melanogaster, where collections of timed and yet somewhat asynchronous embryos are easy to obtain, such that, at least in principle, one can achieve arbitrarily high temporal resolution. Drosophila could therefore serve as a test case to develop a framework for the inference of continuous regulatory and cellular trajectories of in vivo embryogenesis. Because Drosophila is a preeminent model organism that has yielded many advances in the biological and biomedical sciences, obtaining a single-cell atlas of Drosophila embryogenesis is also an important goal in itself. This includes its embryonic development, where the use of this model in conjunction with powerful genetic tools has transformed our understanding of the mechanisms by which developmental complexity is achieved, in addition to uncovering many general principles of both genetic and epigenetic gene regulation.To construct an ungapped representation of embryogenesis in vivo, we would ideally sample embryos continuously. Although this is not practical for most model organisms, it is potentially possible in We profiled chromatin accessibility in almost 1 million nuclei and gene expression in half a million nuclei from eleven overlapping windows spanning the entirety of embryogenesis (0 to 20 hours). To exploit the developmental asynchronicity of embryos from each collection window, we applied deep neural network-based predictive modeling to more-precisely predict the developmental age of each nucleus within the dataset, resulting in continuous, multimodal views of molecular and cellular transitions in absolute time. With these data, the dynamics of enhancer usage and gene expression can be explored within and across lineages at the scale of minutes, including for precise transitions like zygotic genome activation.Drosophila embryonic atlas broadly informs the orchestration of cellular states during the most dynamic stages in the life cycle of metazoan organisms. The inclusion of predicted nuclear ages will facilitate the exploration of the precise time points at which genes become active in distinct tissues as well as how chromatin is remodeled across time.This  Drosophila melanogaster is a powerful, long-standing model for metazoan development and gene regulation. We profiled chromatin accessibility in almost 1 million and gene expression in half a million nuclei from overlapping windows spanning the entirety of embryogenesis. Leveraging developmental asynchronicity within embryo collections, we applied deep neural networks to infer the age of each nucleus, resulting in continuous, multimodal views of molecular and cellular transitions in absolute time. We identify cell lineages; infer their developmental relationships; and link dynamic changes in enhancer usage, transcription factor (TF) expression, and the accessibility of TFs’ cognate motifs. With these data, the dynamics of enhancer usage and gene expression can be explored within and across lineages at the scale of minutes, including for precise transitions like zygotic genome activation. Drosophila embryogenesis.Characterizing the continuum of  We collected staged Drosophila embryos from overlapping time windows across the first 20 hours of embryogenesis. Then we extracted nuclei and performed single-cell RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) profiling using combinatorial indexing (sci-RNA-seq and sci-ATAC-seq) to comprehensively map expressed genes and putatively active regulatory elements. We applied machine learning to infer a continuum of nuclear ages that is synchronized across unfolding lineages in absolute time. The continuous nuclear age predictions were used to annotate and then link cellular states at nonoverlapping 2-hour intervals, as well as to explore transcriptional regulatory dynamics across major cell lineages of embryonic development at fine-scale temporal resolution. Single-cell technologies are a powerful means of studying metazoan development, shedding light on the emergence of cellular diversity and the dynamics of gene regulation. However, nearly all such atlases of embryogenesis are limited in terms of the number of discrete time points and cells sampled per time point. Given the rapidity with which molecular and cellular programs unfold, this limits the resolution at which regulatory transitions can be characterized.Drosophila, where collections of timed and yet somewhat asynchronous embryos are easy to obtain, such that, in principle, one can achieve arbitrarily high temporal resolution. This sharply contrasts with mice, for which there are typically 6 to 24 hours between sampled time points, gaps within which massive molecular and morphological changes take place  and include 85% of known embryonic enhancers, based on overlap with nearly 5000 curated enhancers confirmed in transgenic embryos . –10. Dros embryos , 12, sup embryos . We addi embryos  that are embryos . The com embryos  and 96%  embryos   and fewer identified clusters . We suspHere, we use cell state to mean an annotated cluster at a given time window. Altogether, we identified 171 cell states in sci-ATAC-seq data and 268 in sci-RNA-seq data across the nine time windows, each of which received one of 38 cell type annotations for ATAC or one of 54 cell type annotations for RNA , A and BDrosophila embryogenesis, represented by our 0- to 2-hour time window, include 13 rapid nuclear divisions within a syncytium that generates 6000 nuclei, regulated by maternal genes. At ~2 hours and 20 min after fertilization, cellularization occurs and the zygotic genome is activated  and Delta (Dl), and neuroblast temporal TFs, such as miranda (mira) and castor (cas). Two additional neural progenitor clusters correspond to sensory progenitors, whereas immature neurons express low levels of both neural progenitor and pan-synaptic genes, including cacophony (cac) and synaptotagmin 1 (syt1). Mature neurons are marked by higher levels of pan- and subtype-specific synaptic genes coupled with low or no expression of earlier developmental genes. Finally, midline cells, consisting of both neurons and glia cluster together, become evident at 6 to 8 hours; using the midline TF single minded (sim) and glial immunoglobulin family member wrapper as markers, we can follow them forward in time as they mature (shaven (sv), from 6 to 16 hours  neurons and internal chordotonal (Ch) neurons, and type II multidendritic (MD) neurons. We can clearly distinguish MD neurons on the basis of expression of genes, such as dendritic arbor reduction 1 (dar1), which promotes their characteristic branching dendrites, and the pseudouridine synthase RluA-1, which was recently identified as a marker of MD neurons (pickpocket (ppk) and ppk26. Mechanosensory ES neurons are specified by the TF hamlet (ham), which is specifically expressed in the middle sensory cluster  as well as fate-determinant Rfx and a number of as-yet uncharacterized genes specific to this cluster  and nompA, which promote the development and function of Ch support cells, respectively (ion transport peptide (ITP)], enzymes involved in their synthesis [amontillado (amon)], and receptors [myosuppressin receptor 1 (MsR1)] . Th. ThDroso cluster , 26 ] .complexin (cpx) and CG4328, identified in our analysis as enriched in the monoaminergic cluster, which includes midline neurons  confirmed this unexpected finding  CR31451 as enriched in mature neurons as well as two genes,  neurons . This ne finding  and raisThis deeper exploration of the neuroectoderm, validating and extending years of research from many groups, illustrates the depth of information that can be obtained from these data. We additionally performed a more detailed annotation of nonmyogenic mesoderm . A full eve locus, the stripe 1 enhancer has a much stronger skew for anterior accessibility compared with stripe 2, as has also been previously reported  axis of the blastoderm embryo . Using lreported . Our sinelopment . Using telopment .To further leverage continuous views of unfolding trajectories, we next explored the gene regulatory modules active in germ layer–specific development. We focused on the mesoderm and its derivatives as a complex, well-characterized system that we and others have studied previously , 29, 30.n = 571) are highly expressed from the beginning of mesoderm development ; are enriched for TFs (P = 1.4 × 10−6); and likely represent a mixture of genes involved in progenitor cells, mesoderm development, and transcriptional activation (n = 433) peak at ~9 to 11 hours, during the subdivision of the mesoderm into different muscle primordia and their subsequent specification. This cluster is enriched for genes involved in mesoderm development, including myoblast fusion and myotube differentiation, while losing enrichment for stem cell and self-renewal terms (n = 365) initiate expression at ~10 hours and steadily increase to the end of embryogenesis, whereas cluster 4 genes (n = 631) only switch on at ~15 hours, during muscle terminal differentiation. The last cluster lacks enrichment for TFs and rather includes genes involved in myofibril assembly and muscle assembly and maintenance as well as essential contractile proteins for differentiated muscle , are present in clusters 3 and 4. Mhc protein plays a critical role in providing muscle-contractile force. Our scRNA data show increasing Mhc expression along the muscle lineages in cells with later embryonic ages , a TF associated with muscle development, which has peak expression at 10 hours of embryogenesis  data  data , which spression .q < 1 × 10−3 and presence in >1% of target peaks; hb, en, Ubx, and pb), and concordantly expressed in the first temporal cluster. These factors have many functions, including setting up the segmentation of the mesoderm, regulating the expression of somatic muscle identity genes, establishing midgut constrictions in the visceral mesoderm, and heart patterning. Other examples from the second and third temporal clusters are genes required for cell fate specification of somatic muscle founder cells  and heart development .To extend this analysis more globally, we searched for TF motifs enriched in putative enhancers (mesoderm-specific scATAC peaks 1 to 10 kb upstream of the TSS) of genes belonging to each of the four scRNA mesoderm expression clusters. This identified 458 TF motif-to-cluster enrichments . This caveat notwithstanding, these analyses highlight the potential for further discovery of coregulated gene modules related to distinct germ layers or cell types.We next investigated whether we could leverage the diversity of cell states across embryogenesis to infer which TFs drive specific programs of cell type differentiation. For this, we used all scATAC clusters at all time points  and searched for differential enrichment of TF position weight matrices (PWMs) within each cluster’s open chromatin regions.We first characterized enrichments across clusters from the 10- to 12-hour time window based on predicted time . EncouraBecause members of the same family of TFs typically recognize similar motif sequences , it is often difficult from motif analysis alone to pinpoint the responsible TF. To address this, we leveraged our scRNA data to identify the most likely active TF on the basis of its expression within the clusters among all factors that share the same motif binding pattern. First, we used a regression-based framework to integrate the scATAC and scRNA datasets and identify links between the different cell clusters , 6. Specsage transcript and high accessibility of the Sage-associated PWM  TF-to-tissue relationships having both associated expression and chromatin activity at one or more of the nine time windows assessed. We note that in time windows with fewer clusters, the association effect estimates are susceptible to outliers and should be interpreted with caution. Notwithstanding this caveat, these putative assignments represent an extensive resource for future studies ;  studies .CG5953 and CG11617) or neuroectodermal tissues (Ets65A and CG12605) was poorly characterized. We confirmed that these factors are in fact expressed in the tissue and time window predicted by our data neo38, Lim3, lola, fkh, and fru  besides being positively regulated by the pan-muscle TF Mef2 and repressed by Run and Opa  to developmental age inference. There are cases where technical features of the data can lead to increased uncertainty of model predictions. For example, we found that cells annotated as germ cells, from the first collection time window, or with low read count were associated with greater prediction error . Moving The extensive scATAC data, with deep coverage across almost a million cells, likely captured most regulatory elements active during embryonic development and provides a comprehensive resource of potential enhancers for almost any cell type in the embryo. By contrast, our scRNA data had relatively low unique reads per cell and will likely miss some differentially expressed genes in specific cell types. As a result, some delicate analyses remain challenging. For example, we found transcriptional velocity estimates to be unstable with sparse scRNA data, although this issue was mitigated by constructing metacells before velocity analysis , which mDrosophila embryonic atlas provides broad insights into the orchestration of cellular states during the most dynamic stages in the life cycle of the organism. Our results represent a rich resource for understanding precise time points at which genes become active in distinct tissues as well as how chromatin is remodeled across time. The annotation of cell types within these data is an ongoing process and one that is much more challenging at early and mid-stages of embryogenesis as compared with late time points or in adults with differentiated tissues. A comprehensive annotation of embryonic cell states will require a collective effort from the Drosophila community. To support these ongoing efforts, we provide information on expression and peaks from all clusters  filters for low read depth or high proportions of reads mapping to the mitochondria or ribosomal genes and extensive doublet removal. Between the two data modalities, we obtained profiles for ~1.5 million nuclei, although unique read depth per nucleus was considerably lower for scRNA than scATAC data.A detailed version of the materials and methods is provided in the Using the center hour of the collection window, we used several machine learning approaches to fit a model that could infer the age of a nucleus with either gene expression or chromatin accessibility information. Both LL regression and neural networks were fitted to the same training data, with a held-out subset used for model validation and comparison. Given its consistently superior performance, we then relied on specific parameterizations of NN model–inferred ages to reposition nuclei in time. To zoom into fine-scale time points, we binned data by small increments to explore the regulatory dynamics of ZGA. Then, using 2-hour adjacent windows of cells, we computed clusters of similar cells and performed extensive manual review to annotate each cluster’s likely germ layer and cell type. We then used an iterative approach for constructing an acyclic tree of differentiation by identifying the likely precursor cluster for each cluster in a given time window.Neuroectoderm was iteratively analyzed for deeper annotation of neuronal subtypes, whereas mesoderm was picked for analyses focused on identifying coregulated genes and accessible regions, which were then subjected to ontology and TF motif enrichment analysis. To connect scATAC cell clusters with scRNA cell clusters, we used a regression-based approach (NNLS). Such connections between ATAC and RNA clusters enabled a series of analyses, such as correlating expression with motif accessibility, applying GRN analysis pipelines, etc.Several additional analyses were performed. We used probabilistic label transfer to map likely cluster annotations from these data to spatial information from patterned DNA nanoballs. We also found it is possible to infer the sex of cells from the proportion of chrX-mapped scATAC reads using a Gaussian mixture model to classify cells. Although RNA velocity was challenging to apply to sparse scRNA data, it yielded more sensible results when subsets of cells were first aggregated to metacells.elav with binou, genes active at specific mesoderm time points, and putative active TFs with less-characterized roles in tissue development.The expressions of several genes were verified by fluorescent in situ hybridization: specific neuronal genes active in identified clusters, unexpected coactivity of the Raw data are available through the Gene Expression Omnibus (GEO). Additional scripts and intermediate files, including bigwigs and a custom web app to visualize UMAPs, are available through our data-sharing website.Supplementary materialsSupplementary tables


Memories are lasting representations over time of associations between stimuli or events. In general, the relatively slow consolidation of memories requires protein synthesis with a known exception being the so-called Anesthesia Resistant Memory (ARM) in Drosophila. This protein synthesis-independent memory type survives amnestic shocks after a short, sensitive window post training, and can also emerge after repeated cycles of training in a negatively reinforced olfactory conditioning task, without rest between cycles (massed conditioning—MC). We discussed operational and molecular mechanisms that mediate ARM and differentiate it from protein synthesis-dependent long-term memory (LTM) in Drosophila. Based on the notion that ARM is unlikely to specifically characterize Drosophila, we examined protein synthesis and MC-elicited memories in other species and based on intraspecies shared molecular components and proposed potential relationships of ARM with established memory types in Drosophila and vertebrates. Drosophila melanogaster is capable of multiple forms of aversive and reward learning and memory in both associative and non-associative scenarios. Through its powerful and facile molecular and genetic arsenal, Drosophila has been a cardinal contributor to our understanding of many molecular mechanisms that underlie these processes.Learning and memory are adaptive vital functions that enable animals to predict outcomes based on prior experiences. These outcomes may be rewarding, such as the location of food, shelter, and mates, or beneficial by promoting avoidance of potentially harmful situations, places, and predators. The fruit fly Of the various learning and memory assays, the more robust and well-understood are the associative, negatively, or appetitively reinforced olfactory paradigms. Two equally aversive odorants serve as the conditioned stimuli (CS), while pulses of electric footshocks, or sugarwater are the respective aversive and appetitive unconditioned stimuli (US). One of the two odorants, the CS+, is presented concurrently with the US, while the other (CS-) functions as an unpaired control . LearninLearning in these associative paradigms may be referred to as Immediate Memory (IM), or Short-Term-Memory (STM) because the time interval between training and testing of learning-dependent performance does not typically exceed 3 min. Perdurance of the learned information over time due to processes that maintain and consolidate it constitute memory of the event. The persistence of memory depends on training intensity as well as the nature of the information. For example, one-time association of a specific odor with a food reward leads to much longer-lasting memory than association of the same odor with multiple pairings to electric shock ,4. LaterFor US/CS associations to be maintained over time, processes generally referred to as memory consolidation are activated, probably soon after the learning episode . Memory An effective tool in investigating the time course of memory consolidation in Drosophila was provided by the observation that acute exposure to 4 °C anesthetizes flies, followed by speedy recovery to apparently normally functioning animals minutes after removal from the cold. By definition, anesthetic agents lead to loss of consciousness. Although it is unclear whether the flies are truly anesthetized or simply immobile due to the cold, the treatment was called cold anesthesia. The presumptive linkage probably derives from the analgesic effects of cold, which has been widely used as a local treatment for injuries ,11, and Anesthesia-Resistant Memory (ARM) [Anesthesia-Sensitive Memory (ASM) [The effect of the cold shock is partially alleviated if given 30–40 min post training in one version of the associative conditioning paradigm  and FiguD. By 90–ry (ARM) , while try (ASM) ,6,17. Thradish, presenting selective ARM elimination [Significantly, because it persists the amnestic treatment, ARM must not be labile at that time and therefore it likely represents relatively quickly consolidating memory. In contrast, ASM represents memory of the association which is unconsolidated at that time of cold shock and therefore in a labile phase. Whether ARM and ASM represented the same memory in different stages of consolidation or distinct memories remained an open question until the discovery of one mutant named mination . This armination ,20. The mination ,6 and phmination  becomes Stable protein synthesis-dependent LTM can be achieved by 5–10 cycles of 12 US/CS pairings per cycle of negatively reinforced olfactory conditioning delivered with a 15-minute rest between cycles (Spaced Conditioning-SC) as illustrated in To elicit ARM formation, either of two protocols of aversive olfactory conditioning is typically used. One protocol emulates the original experiment that yields ARM  and invoradish, which disrupts cold-shock insensitive ARM also disrupts MC-induced memory [MC-yielded memory was thought to be anesthesia resistant based on two main points. First, like ARM, MC-induced memory is protein synthesis-independent as it is not susceptible to the protein synthesis inhibitor Cyclohexamide (CXM) , which dd memory . Althougd memory , suggestd memory . WhetherEven though MC-elicited memories, ARM, and LTM partially overlap in time, they differ in their time of emergence, duration, and functional characteristics  gene differentially affect ARM and MC memories, but not LTM [Do ARM and MC memories engage distinct molecular mechanisms than those requisite for LTM? In addition to the protein synthesis independence, evidence for engagement of distinct molecular mechanisms emerged from the abovementioned observation that mutations in the  not LTM ,18,32. T not LTM  and base not LTM . By this not LTM ,40. Thusddc gene encoding the enzyme necessary for serotonin and dopamine biosynthesis also disrupt ARM , an inhibitor of serotonin synthesis, impairs ARM. Of the five serotonin receptors, 5HT1A is essential for ARM formation  and Tabl. In confupt ARM , since a constitutively active form, PKM, enhances ARM independently of Rad . This isdrk), or Growth factor Receptor Bound protein 2 (GRB2) in mammals, present defects both in cold-shock and MC-elicited memories in a Rad-independent manner [A novel molecular pathway engaged in ARM formation was suggested by the observation that mutants in the adaptor protein Downstream of Receptor Kinase , suggesting signaling to actin polymerization/depolymerization. In confirmation of this hypothesis, filamentous actin was significantly reduced in drk mutants and the Drok inhibitor drug Fasudil impaired ARM and decreased filamentous actin levels in the CNS of control flies [Significantly, however, the ARM/MC memory deficit of ol flies . This prol flies , lendingscribble (scrib), whose disruption leads to enhanced ARM, is placed in a pathway downstream of the dopamine receptor Damb and acts through the Rac/Coffilin signaling pathway to mediate forgetting [Additional genes that contribute to ARM/MC memory, with yet unclear roles in the abovementioned or additional molecular pathways are presented in rgetting , again iAnother significant issue that needs to be addressed systematically given the recent finding that MC yields both ARM and PSIM  is to deshibire (tsshi) transgene encoding Dynamin [tsshi transgene in specific neurons blocks neurotransmitter reuptake, essentially synaptically silencing them [What are the neurons in the adult Drosophila CNS essential for ARM? When known, the expression pattern of genes with validated contribution to ARM has led to RNAi-mediated adult-specific knockdown of the encoded proteins therein. Emergence of deficient ARM/MC memory verifies the contribution of these neurons, eliminates the possibility of a developmental origin for the deficit, and ascertains the role of the gene in the process(es). An alternative strategy involves the thermo-sensitive  Dynamin . Acute eing them , enablinrad, drk, and bruchpilot genes are preferentially expressed in the Mushroom Bodies [The m Bodies ,56,57, wm Bodies .ala mutant flies, which lack one or more MBN groups, revealed normal ARM if either α or β neurons were absent but not if both of them were missing [Initial studies on the  missing ,60. Ther missing . The mai missing . On the  missing . The d5H missing . While t missing .dunce appears to be necessary not only in the MBs but also in antennal lobe  local neurons (LNs) for normal ARM [Except for the MBs and their apparent input neurons, other neurons in the fly brain are also involved in ARM/MC memories. The gene rmal ARM . Furtherrmal ARM . ApparenMemory surviving amnestic treatment (ARM) was apparently first described in Drosophila and it is in this experimental organism that the molecular mechanisms and neuronal circuitry that supports it were defined. Although ARM per se has been verified in a couple more species, it is unreasonable to maintain that these elaborate molecular mechanisms and circuitry evolved to serve a particular type of memory specifically in Drosophila. So, are ARM or similar memories present in other species? The collective answer to this question is presented in Aplysia resembles the effects of this type of conditioning in Drosophila, as it elicits short-lasting effects compared with the persistent consequences of SC [Limax flavus. A memory of the aversive association that is resistant to cold-shock emerges if the amnestic treatment is administered not immediately, but within 24 h post-training. Memory past that critical period appears fully consolidated and resistant to the insult [Caenorhabditis elegans yields transcription- and translation-independent memory in contrast to spaced training, which is disrupted by protein synthesis inhibition [C. elegans, MC-yielded memory is sensitive to cold shock administered soon after training in agreement with the abovementioned results in a similar experimental scenario in Drosophila [Operationally, MC of the gill-withdrawal reflex response in the sea slug es of SC . A memore insult . Furtherhibition ,66. Inteosophila .Behavioral assays can be more elaborate in vertebrates than invertebrates because of size and better-understood behavioral repertoires. Nevertheless, as in Drosophila, mammals including humans are amenable to MC, which leads to shorter memory retention than memories elicited by SC. Moreover, in certain behavioral paradigms in mice, such as that of long-term adaptation of the horizontal optokinetic response (HOKR), MC-derived memory is clearly distinct from SC-derived memory in retention duration and dependence on protein synthesis . FurtherIn fear conditioning, rodents are trained to associate an auditory cue with electric foot-shock. The effects of both massed and spaced training on fear memory are in accordance with the findings in Drosophila with respect to increased persistence of memories elicited via spaced than by MC and that SC is more effective for contextual fear conditioning compared with MC. In addition, administration of a protein synthesis inhibitor before fear conditioning in mice affects both the contextual and cued responses of the conditioned animals, irrespective of massed or spaced protocol. Significantly, there appears to be a translation-independent memory trace produced by MC, as shown by Long Term Potentiation (LTP) experiments in mouse hippocampal slices . FurtherHuman studies also suggest differential effects of spaced and massed conditioning on memory, in support of the positive impact of spaced intervals between training sessions on memory retention (reviewed in ref. ). While An important correlation regarding the nature of ARM emerged from studies of molecular mechanisms engaged in amygdala-dependent fear conditioning in rats. These studies revealed a significant common characteristic between ARM in Drosophila and fear memories at the molecular level. Cue-dependent fear conditioning leads to GRB2-mediated signaling to p190 RhoGAP-downstream kinase (ROCK) in Lateral Amygdala (LA) . These arad, drk, drok, or rhoGAP present impaired avoidance memory but normal “safety memory” remains to be experimentally determined. If so, this will provide much-needed insight into the nature of ARM and its relation to vertebrate memories.An additional potential link with fear conditioning in rodents and ARM in Drosophila was provided by a rather impactful recent study. Aversive SC in Drosophila was shown to actually yield two concurrent complementary memories, an avoidance memory of the punished CS+ and a “safety memory” of the unpunished CS-. In fact, the avoidance memory was insensitive to protein synthesis inhibitors, did not depend on spaced conditioning trials, and decayed faster than “safety memory”. Therefore, there are multiple aspects of avoidance memory that resemble ARM and in fact may be linked to fear of the predicted punishment. Whether ARM is at least in part a manifestation related to fear conditioning in vertebrates remains to be determined. Shared components of the molecular cascades involved in Drosophila ARM/MC memories and fear conditioning in rodents are likely to provide initial such correlations. Whether Drosophila mutants in Despite significant advances in elucidating molecular components and neuronal circuitry engaged in ARM, it is apparent that it is still only defined in Drosophila, though it is highly unlikely to operate in this insect alone. Its characteristic translation-independent low energetic cost ,27 and sEstablishment of the anticipated link or correlation to vertebrate memories will broaden the scope of ARM/MC memories research. Drosophila, with its extensive molecular and genetic arsenal and continuously enriched behavioral repertoire, is still uniquely poised to reveal novel genes, signaling pathways, cellular mechanisms, and neuronal circuits underlying ARM. Such information will likely prove invaluable towards understanding this novel memory type in vertebrates and reveal potential links to human pathologies affecting cognition.


MYCN amplification, have been associated with adverse outcomes. Using whole transcriptome sequencing, we previously explored the immune microenvironment of neuroblastoma subtypes and discovered its association with clinical outcome. Specifically, we found that patients with tumors infiltrated by higher levels of cytotoxic lymphocytes had a better overall survival. Additionally, we found that a high MYCN gene expression signature in MYCN-non-amplified tumors is an independent predictor of adverse outcome. However, signatures of tumor infiltrating cytotoxic immune cells in this subtype of tumors predict an improved outcome. While this is clinically informative, it does not provide a full picture of the dynamics underlying the biology of tumor immune microenvironment and how to use this information to improve patient outcomes. Here, we highlight our previous work and current approaches using immunotherapy in neuroblastoma and explore our current understanding of the immune biology of these tumors. We further describe how this correlates with patient outcome, and how this information can be used to develop novel immunotherapeutic strategies for pediatric patients with neuroblastoma.Pediatric neuroblastoma is a heterogenous disease that accounts for significant morbidity and mortality in children. Deep genomic and transcriptomic profiling of patient tumors has revealed a low mutational burden and a paucity of therapeutic targets. Furthermore, different molecular subtypes, such as  MYCN-amplified (MYCN-A) tumors or a diagnosis over the age of 18 months with metastatic disease, remains lower than 50%. Additionally, there is significant morbidity associated with the aggressive multimodal therapies used in this patient population. Therefore, the development of targeted therapies with lower toxicities is imperative.Neuroblastoma is derived from the developing sympathetic nervous system and is the most common extracranial solid tumor of childhood. It is typically diagnosed within the first two years of life and accounts for approximately 15% of all pediatric cancer deaths . The cliMYCN-not-amplified (MYCNNA) neuroblastomas have increased T-cell infiltration, suggesting the presence of antigenic targets and an active anti-tumor immune response .,22.MYCN-MYCN-NA tumors had significantly higher scores compared to MYCN-A tumors  clones recognizing tumor specific antigens. We found that the number of total TCR clones was correlated with both the CD8+ T-cell score and overall immune scores across all samples , are targets of FDA approved therapeutics, which may have the potential to improve outcomes in this high-risk neuroblastoma patient population.T- and NK-cell exhaustion can allow tumors to evade killing by the patient’s immune system ,27. In oactivity . InteresIn summary, our findings of prognostic immune signatures including cytotoxic (T and NK) cell activation, expansion of TCR clones, cytolytic signatures, upregulation of immunosuppressive markers, indicates that the TILs may be capable of specifically targeting neuroblastoma cells. This may be harnessed for combinatorial immunotherapeutic approaches, which could improve patient outcomes. Overall, our study demonstrates a strong correlation between the neuroblastoma TME and patient outcome.MYCN-A tumors, which are characterized by a “cold” TME with few immune cell infiltrates .Immune checkpoint blockade (ICB), which utilizes mAbs targeting molecules known to dampen anti-tumor immune responses, has shown dramatic results for adult patients with solid tumors, and agents targeting the immune checkpoints CTLA-4 and PD-1 have been approved by the FDA . IpilimuMore recently, adoptive cell therapies (ACTs) have been trialed to treat neuroblastoma. The majority of these are using CAR T-cells to target surface antigens such as GD2, CD171, CD276, or EGFR, and there are a total of 18 completed or ongoing CAR T-cell trials treating neuroblastoma patients  43–46].–46.43–46Our group and others have shown that there are different subtypes of TMEs found in neuroblastoma and that the TME of pediatric neuroblastoma is not only associated but plays an active role in the clinical outcomes of these patients ,12,50,51We hypothesize that response to mAbs targeting TAAs in neuroblastoma may be predicted by the presence of cytotoxic cells, such as NK-cells and T-cells in the TME, such as those seen in Cluster 3 and Cluster 4a. There are several mechanisms of action of mAbs, but many are dependent on cytotoxic innate or adaptive immune cells. For example, they can directly activate complement mediated cytotoxicity or their effects can be indirect through antibody-dependent cellular cytotoxicity (ADCC) . When GDMany neuroblastomas lack tumor infiltrating lymphocytes (TILs) in their TMEs, such as tumors categorized as Cluster 1 or Cluster 4b. One of the causes can be attributed to low immunogenicity of tumor subtypes. It has been shown that increased expression of MYCN through amplification or its functional gene signature can downregulate MHC Class I on the cell surface, which cells use to present antigens to T-cells –25. StraOne established mechanism of resistance to ACT is immune cell exhaustion –65. ExhaBesides cytotoxic immune cells, other components of the TME, such as cytokines and immunosuppressive cells, have been associated with clinical outcomes and identified as sources for sub-optimal ACT activity. Therefore, modulating the immunosuppressive elements of the TME could improve therapeutic responses. TGF-β has been implicated in suppressing cytotoxicity and infiltration of both NK-cells and T-cells –73. TargNeuroblastoma displays four recurrent patterns in the composition of the TME. These patterns are associated with distinct patient outcomes and may indicate tumor susceptibilities to specific immunotherapies. Currently, predicting responses to immunotherapies remains challenging, and since their mechanisms of action are dependent on the interplay between a patient’s tumor and immune system, understanding the TMEs of individual tumors could provide clinical rationale for selecting therapeutic approaches. Furthermore, as the TME can evolve over time under therapeutic pressures, evaluation of these changes in the context of exposure to therapy to better understand therapeutic effects on the TME and to optimize the timing and choice of cancer treatments including immunotherapeutic strategies for these patients.In summary, neuroblastoma transcriptome profiling has revealed notable characteristics of the TME including immune infiltrates in high-risk neuroblastoma and their relationship to patient outcome. These results warrant further studies, including spatial transcriptomics or proteomics to validate and develop a deeper understanding of the interplay between tumor cells and TME. Only with this knowledge, biomarkers based on tumor and TME phenotypes can then be identified to guide patient stratification and therapeutic strategies to improve the outcomes for patients with high-risk neuroblastoma.JCI-21-112_Supplementary file


Memory disturbances are amongst the most common and disruptive symptoms of chemotherapy-related cognitive impairment. Chemotherapy treatments commonly cause neurotoxicity within the hippocampus, creating a vulnerability to memory impairment. Most clinical assessments of long-term memory in breast cancer survivors assess basic verbal and visual memory processing, and do not capture the complexities of everyday event memories, including episodic and autobiographical memory. This review focuses on structural and functional neuroimaging studies identifying disruptions in the hippocampus and recollection network, and related episodic memory impairments in chemotherapy-treated breast cancer survivors. We argue for the need to better characterize memory dysfunction following chemotherapy treatments. Given the importance of episodic and autobiographical memory to a person’s personal history and quality of life, an under-appreciation of how this memory domain is impacted by standard cancer treatments potentially diminishes the negative experiences of breast cancer survivors, and neglects cognitive problems that could benefit from intervention strategies.Long-term memory disturbances are amongst the most common and disruptive cognitive symptoms experienced by breast cancer survivors following chemotherapy. To date, most clinical assessments of long-term memory dysfunction in breast cancer survivors have utilized basic verbal and visual memory tasks that do not capture the complexities of everyday event memories. Complex event memories, including episodic memory and autobiographical memory, critically rely on hippocampal processing for encoding and retrieval. Systemic chemotherapy treatments used in breast cancer commonly cause neurotoxicity within the hippocampus, thereby creating a vulnerability to memory impairment. We review structural and functional neuroimaging studies that have identified disruptions in the recollection network and related episodic memory impairments in chemotherapy-treated breast cancer survivors, and argue for the need to better characterize hippocampally mediated memory dysfunction following chemotherapy treatments. Given the importance of autobiographical memory for a person’s sense of identity, ability to plan for the future, and general functioning, under-appreciation of how this type of memory is impacted by cancer treatment can lead to overlooking or minimizing the negative experiences of breast cancer survivors, and neglecting a cognitive domain that may benefit from intervention strategies. Advances in diagnostic and therapeutic interventions for breast cancer have led to high patient survival rates . The retCRCI has been seen in many other types of non-CNS cancers  but is mWe searched the PubMed and Google Scholar data bases between 2000 and 2022 using the search terms ‘chemotherapy’, ‘breast cancer’, ‘chemofog’, ‘chemotherapy-induced cognitive impairment’, ‘episodic memory’ ‘long-term memory’, ‘autobiographical memory’, ‘resting state’, ‘default mode network’, ‘hippocampus’, ‘temporal lobe’, ‘MRI,’ ‘fMRI’. Papers were excluded if they did not include measures of long-term memory  or structural or functional assessments of the temporal lobes or recollection/default mode network in chemotherapy-treated breast cancer survivors.neurogenesis. The hippocampus, a medial-temporal lobe structure that is critical for memory processing, has been found to be particularly sensitive to structural and functional disruption following chemotherapy treatment . The phyHippocampal subregions  have specialized functions, with the dentate gyrus being of particular interest in understanding chemotherapy-related cognitive impairment and neurotoxicity due to its role in neurogenesis. The dentate gyrus is unique, in that it is one of two known regions to continually generate new neurons in the mammalian brain . This prSeveral molecular mechanisms referred to above have been linked to reduced rates of hippocampal neurogenesis. For example, cell damage induced by chemotherapy-induced increases in reactive oxidative stress reduces the survival of primary neural precursor cells and inhibits the production of new cells in the hippocampus ,25,42,43The range of systemic chemotherapeutic agents commonly used in breast cancer therapy have been shown to suppress hippocampal neurogenesis and to impair hippocampally mediated memory in rodents, including the anti-metabolites methotrexate ,46,47,48In vivo structural neuroimaging studies in humans lack the spatial resolution to assess differences in dentate gyrus volume at the cellular level, and cannot distinguish natally generated neurons from adult-generated neurons. However, hippocampal segmentation analyses have identified differences in hippocampal sub-region volume between chemotherapy-treated breast cancer survivors and healthy controls, suggesting that systemic chemotherapy treatments are capable of inducing changes in the human hippocampal architecture . Post-moGiven the role of the hippocampus in memory processing, understanding changes in hippocampal integrity following various chemotherapy treatments is essential to understanding the associated memory impairments in breast cancer survivors. Quantifying adult hippocampal neurogenesis remains a challenge in humans ,76. TherMuch of what is known about CRCI and its underlying cellular and molecular mechanisms has been identified in pre-clinical studies of rodents ,31,72,82California Verbal Learning Test and the Brief Visuospatial Memory Test-Revised, for example, have identified lower scores relative to pre-treatment baseline and to healthy controls, persisting up to one year post-chemotherapy [This limitation in test complexity is not unique to pre-clinical measures. The most common neurocognitive tests of long-term memory performance in breast cancer survivors measure verbal and visual memory using standardized list learning, word or object recognition, or free recall tasks following a delay. These are well established tasks that are sensitive to mild cognitive impairment associated with hippocampal impairment ,84,85. Gotherapy ,89,90. Sepisodic memory employ paired associates learning tasks at encoding, requiring participants to later recognize paired items, words, or spatial contexts, presented during encoding and again during a recognition test [declarative memory processing required to support the encoding (formation) and recollection of real-life, everyday events and the related semantic information that is an intricate part of human memory for personal experiences [Other neurocognitive assessments of ion test ,91. Epision test ,93,94. Uion test . See ‘Neeriences .Declarative memory, or memory that can be voluntarily called into consciousness, is comprised of both episodic and semantic elements . Semantischematic features of the event , and semantic information related to the event ”. Despite this normal loss of memory for episodic details, if given a salient cue at the time of retrieval, healthy individuals can probably access those precise episodic details even after a very long time .Even in healthy individuals, the precise episodic elements of a memory are susceptible to forgetting over time whereas the semantic elements of a memory tend to be more stable ,101,102.Trace Transformation Theory, which posits that episodic memories are consolidated in rich contextual and perceptual detail within the hippocampus. So long as the memory remains accessible, the hippocampus continues to be required for the retrieval of contextually and perceptually detailed elements of the memory. Over time, episodic memories are transformed into less detailed schematic memories that capture the essential features or gist of the original event. Storage and retrieval of this form of the memory are supported by neocortical regions, with the prefrontal cortex playing a particularly important role. The detailed hippocampus-dependent version, and the transformed schematic version of the memory, can co-exist in the healthy brain, and the available cues at the time of retrieval will direct which version is retrieved [Observations of differential loss of memory details can be accounted for by the etrieved ,95,120. etrieved ,122.Given this profile of memory consolidation, it is probable that even subtle hippocampal disruption, such as that resulting from chemotherapy-induced neurogenic suppression or hippocampal atrophy would result in selective impairment in the retrieval of episodic memories. Such results have been identified in preclinical rodent models in which adult hippocampal neurogenesis has been ablated using chemotherapy ,61 or crAutobiographical memory is a unique form of declarative memory that unfolds over time, involves temporal and spatial sequencing of an event, and is comprised of a complex interaction of episodic and semantic elements of a personally experienced event. To recall an autobiographical memory, details that must be accessed from memory stores, reconstructed and elaborated upon during the retrieval process [retrograde memories that are preserved in the presence of hippocampal damage, however, tend to lack episodic specificity, and rather retain a more gist-like and semantic version of the event [ process ,126. Pathe event ,127,128.Despite the pervasive reports of memory impairment in those experiencing CRCI and the known susceptibility of the hippocampus to the neurotoxic effects of chemotherapy, only a limited number of studies have investigated autobiographical memory processing in breast cancer survivors. One early investigation using the Autobiographical Memory Task found that Ch+ breast cancer survivors demonstrated reduced ability to produce ‘specific’ autobiographical memories in response to positive, negative, or neutrally valenced cue words when compared with heathy controls . Rather TEMPau task, a semi-structured interview that assesses memories for unique events occurring at specific times and places within three lifetime periods: the five years before treatment, the cancer treatment period, and the 12 month post-treatment period [Another study assessed both pre- and post-treatment autobiographical memories in breast cancer survivors using the more rigorous t period . The rest period ,133,134,Autobiographical Interview [Several tests have been developed to probe the qualitative content of autobiographical memories, including the Autobiographical Memory Interview  and the nterview . The Autrecollection network  breast cancer survivors, failed to find any difference in hippocampal volume, whole brain volume, or any differences in delayed verbal of visual memory performance between Ch+ and Ch− survivors . Given tAccordingly, structural imaging of both Ch+ and Ch− breast cancer survivors approximately one year following surgery and chemotherapy treatment identified smaller gray matter and white matter volumes within the parahippocampus, adjacent to the hippocampus, and recollection network regions including the prefrontal cortex and precuneus in Ch+ survivors . SmallerSince these early studies highlighting the need for a more longitudinal approach to monitoring chemotherapy-induced neural alterations, many subsequent MRI studies of brain volume and morphology have identified structural differences in white matter tracts and gray matter volumes across various brain regions in Ch+ breast cancer survivors ,145,146.voxel-based morphometry (VBM) prior to, and following, chemotherapy treatment found significantly lower gray matter volumes within the bilateral hippocampus, parahippocampus, superior temporal gyrus, and regions in the frontal lobes, cerebellum, and thalamus just one month following treatment  has connections with the ventromedial prefrontal cortex (vmPFC), and is associated with the processing of schematic memories ,147. TheIn their study of autobiographical memory in breast cancer survivors 18 months post-treatment, Bergouignan and colleagues (2011) observed a specific deficit in recalling temporal details within autobiographical memory . They alAn early study of fMRI neural dynamics and memory performance identified significantly greater activity across broad regions of the recollection network  in Ch+ bWhile network hyperactivity may reflect a compensatory response supporting memory performance following chemotherapy treatment, network hypoactivity has been associated with poor memory performance. A study of fMRI neural dynamics in Ch+ and Ch− breast cancer survivors conducted ten years after the completion of a high-dosage chemotherapy treatment found long-lasting hypoactivation of the parahippocampal gyrus in Ch+ survivors during encoding of a paired associates episodic memory task in which participants were shown a series of combinations of faces and contexts . During a subsequent recognition task in which participants had to judge the accuracy of the face-context pairings after a delay of several minutes, Ch+ breast cancer survivors had lower recognition accuracy scores than Ch− survivors . This stSubsequent longitudinal investigation using this same paired associates task in Ch+, Ch−, and healthy controls  identifiAnother study involving longitudinal tracking of functional networks in breast cancer survivors identified chemotherapy-induced changes in a widespread network of regions while performing a verbal memory task one month following chemotherapy . Relativimplicit memory (non-declarative memory) for the task. Reduced eye-movement discrimination was associated with hippocampal hypoactivation and smaller hippocampal volume, compared to control levels [functional connectivity analyses of these data, Apple and colleagues (2018) identified strong intra-hippocampal connectivity for both Ch+ patients and controls, but Ch+ patients showed evidence of enhanced hippocampal connectivity with the left cuneus and precuneus, lingual gyrus, and right middle frontal gyrus compared to healthy control levels. Higher hippocampal connectivity with the precuneus was associated with higher reports of subjective cognitive concern scores in Ch+ patients, suggesting that hyper-connectivity within these nodes of the recollection network may be compensatory in supporting normal memory performance and needed to overcome anxiety-induced behavioural deficits associated with subjective concern over one’s cognitive abilities [A preliminary study investigated eye-tracking during fMRI scanning in Ch+ patients and healthy controls while they performed the Picture Sequence Memory Test, an established spatial recognition memory task that is sensitive to hippocampal dysfunction . While Cl levels . The conl levels ,156,157.bilities .default mode network (DMN). The DMN is a collection of brain regions that are active when engaged in passive, internally focused cognition (mind wandering) or during a resting state [constructive episodic simulation’ [Many of the neural regions identified as comprising the recollection network overlap with nodes of the ng state ,161,162.ng state ,163. Neung state ,166,167.ulation’ ,168,169.Episodic future thinking is a complex form of prospective thinking which involves imagining or mentally projecting oneself into the future in order to pre-experience events. This process relies on similar cognitive processing and neural network activation involved in episodic recollection [Prospective memory is a form of future thinking that involves planning and remembering to execute a task in the future . It is mllection ,177,178.llection ,180,181,It is unsurprising that individuals with damage or dysfunction within the MTL, including key nodes of the recollection and DMN networks, engage in episodically impoverished mind-wandering. Mind wandering occurs in individuals with MTL disruption, but unlike healthy adults who report thoughts and recollections about the past, and future imagining during mind wandering, individuals with MTL damage report more semantically based thoughts about the present. This reflects an inability to engage hippocampally-mediated recollective processing required for episodic memory retrieval or episodic future thinking . Bruno and colleagues  were themulti-voxel pattern analyses (MVPA) of 19 seed regions within the DMN during a resting task, Kesler et al. (2013) were able to distinguish Ch+ breast cancer survivors from Ch−, from healthy controls with a high degree of accuracy [Modelling analyses by Kesler and colleagues (2017) found that pre-treatment resting state network dynamics can be used to predict the development of cognitive impairment in the first year following chemotherapy treatment, suggesting that irregularities in network dynamics are already detectable at the time of disease onset, and are further exacerbated by chemotherapy treatment . Using maccuracy . MVPA isaccuracy . MVPA waSeed-based connectivity analyses during a resting state task by Cheng and colleagues  identifiPost-treatment perturbations in DMN connectivity patterns have been proposed as a potential biomarker of chemotherapy-induced neurotoxicity, and assessment of patient’s resting state network dynamics may be a useful non-invasive diagnostic tool for identifying those requiring cognitive intervention post-treatment . While tFatigue, anxiety, stress, and other psychosocial factors likely influence cognitive performance, and confound interpretations of performance on standard neurocognitive tests ,113,190.Self-perceived impairments in memory are a common complaint following chemotherapy treatment ,146,193.Following a cancer diagnosis, approximately 14% of patients develop cancer-related post-traumatic stress disorder ,200. TheThis review and commentary on the current state of the memory-related literature in the field of CRCI has identified a gap in our knowledge of the impact of chemotherapy on complex episodic memory processing and alterations to the recollection network. In light of the susceptibility of the hippocampus to chemotherapy-induced neurotoxicity, and the critical role of the hippocampus in episodic memory processing, it is surprising that there has been so little investigation of complex event and autobiographical memory processing in cases of CRCI. A multidisciplinary approach that combines complementary assessments of lab-based neurocognitive episodic memory performance, with more complex real-life event memory assessments  is needed to fully characterize the specific memory domains affected by cancer onset and chemotherapy treatments.While chemotherapy-related suppression of neurogenesis is a leading candidate underlying the memory disruptions and hippocampal functional impairment, other physiological factors likely also contribute to these deficits, including white-matter degradation in the hippocampus and throughout other regions of the recollection network , pro-infInvestigation of these cognitive domains in CRCI are still in their infancy. Given the importance of autobiographical memory to a person’s personal history, sense of identity, and ability to plan for the future, an under-appreciation of how this memory domain may be impaired by standard cancer treatments, has the effect of diminishing the negative experiences of breast cancer survivors, and neglecting cognitive problems that could benefit from intervention strategies.


Enterococci (VRE) genes are bacteria strains generated from Gram-positive bacteria and resistant to one of the glycopeptides antibiotics, commonly, vancomycin. VRE genes have been identified worldwide and exhibit considerable phenotypic and genotypic variations. There are six identified phenotypes of vancomycin-resistant genes: VanA, VanB, VanC, VanD, VanE, and VanG. The VanA and VanB strains are often found in the clinical laboratory because they are very resistant to vancomycin. VanA bacteria can pose significant issues for hospitalized patients due to their ability to spread to other Gram-positive infections, which changes their genetic material to increase their resistance to the antibiotics used during treatment. This review summarizes the established methods for detecting VRE strains utilizing traditional, immunoassay, and molecular approaches and then focuses on potential electrochemical DNA biosensors to be developed. However, from the literature search, no information was reported on developing electrochemical biosensors for detecting VRE genes; only the electrochemical detection of vancomycin-susceptible bacteria was reported. Thus, strategies to create robust, selective, and miniaturized electrochemical DNA biosensor platforms to detect VRE genes are also discussed.Vancomycin-resistant  Enterococci (VRE) bacteria have been a primary nosocomial infection globally, and it is commonly spread via poor hospital hygiene practice. This contributes to higher treatment costs, more extended hospital stays, and a higher death rate 3−/4− electrochemistry on the electrode surface, as shown in 6)]3−/4− and the negatively 3−/4− charged phosphate of DNA strands strongly repel each other, which impedes [Fe(CN6)]3−/4− electrochemistry even more. This method is also straightforward to implement; however, when employing voltammetric techniques such as CV, DPV, and SQW to detect a very small DNA target concentration, the electrochemical signals from the inhibition of [Fe(CN6)]3−/4− are not that significant in terms of decreased current. Hence, to make a more sensitive measurement, EIS is the best electrochemical technique, as the EIS is very sensitive to monitor any changes on the electrode surface, particularly the formation of a double-helix DNA structure. As a result, the hybridized dsDNA structures significantly decrease the conductivity and increase the surface resistance of the modified electrode. In a label-free impedimetric measurement, a ferrocyanide and ferricyanide solution with an equimolar is used. The detection response is monitored through Nyquist plots, where the charge transfer resistance (Rct) can be determined. The increase in the semicircle diameter of the Nyquist plot is directly proportional to the Rct value. This signifies that the hybridized dsDNA has been formed on the electrode surface. Nevertheless, analyzing the data for the calibration plot will not be a straightforward analysis as the Rct value needs to reference the equivalent circuit model representing the electrical response of the electrochemical interface. Consequently, this approach requires skilled personnel who understand the physiochemical processes [Fe(CN6)]3−/4− electrochemistry at the electrode/electrolyte interface.The second approach is monitoring the decreased current from the ferrocyanide or ferricyanide solution through voltammetric measurements. The decreased current is due to the blocking effect of the hybridized dsDNA structure towards [Fe and gold nanoparticles, via coated films that provide nanoplatforms from ssDNA molecule immobilization. The utilization of nanomaterials can increase the electroactive surface area . Thus, mAfter the electrochemical DNA biosensors for VRE genes were successfully developed, a crucial part of our study was testing the developed DNA biosensors on real biological samples to confirm the VRE infections. Hence, the VRE genes were extracted from Gram-positive-bacteria in various real human samples such as infected wounds, serum, stool, and urine. Therefore, the extraction of DNA for real sample measurements is a vital process of this study. A typical and common challenge confronted by biosensors developed for application to real samples is the matrix of the specimen samples, which may likely interfere with the results or negatively affect the biosensor’s detecting principle. Several considerations, such as the sample type, the amount of sample available, the cost of the extraction methods, and the time it takes to extract the DNA, influence the decision of which DNA extraction method to use. Liao and his group have described the difficulties involved with the sample preparation required for diagnostic testing for clinical specimens such as blood , stool, urine, tissue, sputum, and saliva . There aConventional extraction methods, as in ® DNA Isolation Kit (MoBio-K). Although the Ph/Chl + C3 approach took less time, there were some health and safety problems with phenol and chloroform exposure and disposal compared to MoBio-K, which is simple and straightforwardly applied on the benchtop with standard laboratory safety measures [As previously mentioned, the conventional method of extracting DNA has drawbacks; therefore, several strategies for rapid DNA extraction and more affordable methods have been reported. A variety of commercial DNA extraction kits used are shown in measures .It is essential to extract DNA in high quantities and quality to improve the DNA extraction method. DNA extraction using magnetic nanoparticles (MNPs) is widely recognized as a powerful technique. MNPs have a high surface-area-to-volume ratio, a high binding rate with detecting compounds, and the ability to perform magnetically regulated aggregation and dispersion. Because of their high dispersibility, MNPs are also able to rapidly and efficiently bind to biomolecules. Chen et al. studied MNPs and used them to develop an automatic quick nucleic acid extractor that can process 16 samples at the same time. The nucleic acid extraction method can perform an entire extraction in 30 min and is reliable and stable . A magneIn summary, the optimal DNA extraction method of real samples for electrochemical DNA biosensors should meet the following criteria: being simple, sensitive, rapid, and efficient. DNA extraction techniques should also take the following into consideration: high DNA recoveries, impurity and inhibitor removals, and high-throughput processing.Conventional detection methods for VRE genes have been commonly used in hospital clinical laboratories. However, the detection costs are still too high, especially in low- and medium-income countries. Consequently, reducing detection costs and simplifying detection systems have always been the primary goals in industrial and commercialization detection. The development of electrochemical DNA biosensors can replace one of the most traditional methods since interest in bacteria detection biosensing technology-based electrochemical measurements is rising. Conventional gold (Au) and glassy carbon (GC) electrodes are commonly utilized in developing electrochemical DNA biosensors for bacterial detection in which a three-electrode system  is manually set up. Nevertheless, when using screen-printed electrodes (SPEs), such as screen-printed carbon electrodes (SPCEs) and screen-printed gold electrodes (SPGEs), measurements are easy because the three-electrode system is on the same electrode platform. This makes them an attractive disposable sensor for detecting VRE genes. Screen-printed electrodes offer numerous advantages, including low cost, design flexibility, high reproducibility, microvolumes of samples used, and a wide range of options for modifying the properties of electrode surfaces. During hospital outbreaks, it is important and necessary to reduce the time needed to diagnose VRE. Thus, using electrochemical DNA biosensors to detect VRE genes-based SPEs have a significant potential for making high-selectivity detection systems by creating DNA platforms that can only detect sequence-specific activity towards the VRE genes of interest. Additionally, the modified electrodes allow for rapid in situ analysis with high reproducibility, sensitivity, and accuracy.Recent advances in 3D printing technology and the availability of many conductive thermoplastic filaments to produce 3D-printed electrodes have enabled the additive manufacturing of 3D biosensing devices. Typically, this is accomplished by printing the electrodes with various conductive thermoplastic carbon nanomaterials, which are a composite of the polylactic acid (PLA) polymer and carbon nanomaterials  with a range of designs and specified geometries . HoweverEven though DNA biosensors are often utilized in electroanalysis, extensive data display platforms are still required for signal output. This flaw not only raises detection costs but also affects the growth of on-site detection. Due to the widespread use of smartphones, many studies have developed applications that generate detection signals. The smartphone’s high-quality camera, portability, and feasibility make it a great development candidate. Additionally, smartphones can be used as reading devices. It may become a research focus in the future for the development of miniaturized and portable electrochemical DNA biosensors for detecting VRE genes.


Such volumetric imaging tools provide the opportunity to assess the heterogeneity of airway remodeling within the whole lung and have led to the novel identification of heterogenous gas trapping and mucus plugging as important predictors of patient outcomes. Lastly, we summarize the current knowledge of modification of airway remodeling with available asthma therapeutics to highlight the need for future studies that use in vivo imaging tools to assess airway remodeling outcomes.Asthma affects an estimated 262 million people worldwide and caused over 461,000 deaths in 2019. The disease is characterized by chronic airway inflammation, reversible bronchoconstriction, and airway remodeling. Longitudinal studies have shown that current treatments for asthma  can reduce the frequency of exacerbations, but do not modify disease outcomes over time. Further, longitudinal studies in children to adulthood have shown that these treatments do not improve asthma severity or fixed airflow obstruction over time. In asthma, fixed airflow obstruction is caused by remodeling of the airway wall, but such airway remodeling also significantly contributes to airway closure during bronchoconstriction in acute asthmatic episodes. The goal of the current review is to understand what is known about the heterogeneity of airway remodeling in asthma and how this contributes to the disease process. We provide an overview of the existing knowledge on airway remodeling features observed in asthma, including loss of epithelial integrity, mucous cell metaplasia, extracellular matrix remodeling in both the airways and vessels, angiogenesis, and increased smooth muscle mass. While such studies have provided extensive knowledge on different aspects of airway remodeling, they have relied on biopsy sampling or pathological assessment of lungs from fatal asthma patients, which have limitations for understanding airway heterogeneity and the entire asthma syndrome. To further understand the heterogeneity of airway remodeling in asthma, we highlight the potential of  Lastly, we review current therapeutics and their effect on features of airway remodeling.Asthma affects an estimated 262 million people worldwide and caused over 461,000 deaths in 2019 . The disTo further understand asthma pathology, the field has focused on clinical asthma phenotypes which have been generated from recognizable clusters of demographic, clinical, or pathophysiological characteristics of patients with asthma. Some of the most common asthma phenotypes are early-onset or late-onset allergic asthma, non-allergic asthma, smoking-related, obesity-related, or aspirin-exacerbated respiratory disease (AERD) . Most reTo date, endotyping the inflammatory profile within a patient with asthma has been important clinically to understand which patients will be the most likely to respond to inhaled corticosteroids or who will benefit from the treatment with biologics. However, independent of the asthma phenotype or endotype, the underlying pathologies of asthma are chronic inflammation, reversible bronchoconstriction, and airway remodeling. In asthma, airway remodeling of the conducting airway walls results in fixed airflow obstruction, but airway remodeling also significantly contributes to airway closure during bronchoconstriction during an acute asthmatic episode . SeveralAirway remodeling was first described in cases of fatal asthma by Abnormal thickening of the reticular basement membrane with increased deposition of extracellular matrix (ECM) proteins, fibronectin, collagen types I, III, and V, hyaluronan, laminin α2/β2, tenascin, and versican within the lamina reticularis is one of the hallmarks of airway remodeling in asthma and has been observed in both children and adults with mild to severe and fatal asthma in several histological studies . SeveralIncreased deposition of collagen I, collagen III, and fibronectin have also been demonstrated in the lamina propria of patients with asthma . FibroblLastly, respiratory viruses may also play a role in increased ECM remodeling in asthma. Rhinovirus (RV) infection of airway smooth muscle cells increases the deposition of ECM proteins fibronectin, perlecan, and collagen IV . FurtherIn summary, basement membrane thickening is an early and universal feature of airway wall remodeling in asthma. It has been proposed that basement membrane thickening may lead to loss of epithelial-fibroblast cross-talk leading to subepithelial fibrosis. Indeed Osei et al. have shown that epithelial-derived interleukin (IL)-1α is an important mediator in epithelial-fibroblast cross-talk and reduces inflammatory cytokine release and ECM production in healthy lung-derived fibroblasts . Whethervia the release of alarmins   compared to healthy controls  . Indeed ression) , and ex controls . In respcontrols . Whereascontrols .In situ studies have also revealed many structural changes within the airway epithelium of asthmatic patients, including disruption of tight junctions and adherens junctions, detachment of ciliated cells, increased numbers of goblet cells, basal cells (CK5+/p63+), and side population progenitor cells (PCDH1 (protocadherin-1) (ORMDL3 (orosomucoid-like 3) (DPP10 (dipeptidyl peptidase 10) (GPRA (G protein-coupled receptor for asthma susceptibility) (CDHR3 (cadherin-related family member 3)  , ORMDL3 -like 3) , DPP10 (dase 10) , GPRA (Gibility)  and CDHRember 3) . CDHR3, nfection . Althougal cells . It is tal cells .AR (Goblet cell hyperplasia (increased number of cells), metaplasia (change in cell phenotype) and mucus accumulation have been consistently reported in the conducting airways of patients with mild, moderate, severe, and fatal asthma . In the AR . It has AR .via mucociliary clearance  hyperresponsiveness is a key feature of acute airflow limitation in asthma and its mechanisms of contraction and relaxation have been a subject of great investigation . Increasreceptor . Furtherreceptor . These creceptor . Lambertreceptor . This noreceptor . During receptor . Johnsonreceptor . The defreceptor . This sureceptor . Togethereceptor .It has been previously shown that placing a load on healthy airways stretches the ASM more than the optimal length, causing them to produce less contractile force . HoweverMore recently the role of ASM in asthma pathology has been shown to extend beyond bronchoconstriction. There is now compelling evidence that ASM is involved in airway inflammation through the release of inflammatory chemokines IL-1β, TNFα or immunomodulators including IL-5, IL-13, TGF-β . ASM addIn summary, the contribution of ASM in asthma is multifactorial and beyond the effects of bronchoconstriction alone. ASM contraction not only alters the mechanical properties of the airway wall but also contributes to airway remodeling which influences other structural cells of the airway  and immune modulation (inflammatory cells).While the histological studies described above have provided extensive knowledge on different aspects of airway remodeling in asthma, they have relied on biopsy sampling or autopsy lungs which have several limitations to note. Biopsy samples are limited in the airway wall structures that can be sampled which are primarily the epithelium, basement membrane and laminar reticularis, and the tissue artefacts that can be induced when crunched by the biopsy forceps. Additionally, lung biopsies primarily allow for the larger airways to be sampled and it is the branch point of two daughter airway generations that is sampled which can make sampling of the airway wall very difficult. Lastly, it is very difficult to sample multiple airway generations to assess the heterogeneity of airway remodeling within the lung of asthmatic patients. Autopsy studies on lungs from fatal asthmatics offer a unique opportunity to assess the heterogeneity of airway remodeling within multiple airway generations and across lung height, however, it does not provide the opportunity to assess the spectrum of the asthma syndrome specifically mild and moderate asthma. Below we describe the various approaches that have been used to assess the heterogeneity of airway remodeling and its effects on lung function. The inherent heterogeneity of lung ventilation has long been of great interest, dating back to 1956 when the uneven distribution of ventilation was hypothesized to influence the mechanical properties of the dynamic lung . This hyin vivo in patients with asthma. This has overcome many limitations of histological studies which could only be conducted on lungs from fatal asthma patients, and the lack of airway wall structures sampled in an airway wall biopsy sample. Over the past three decades, numerous studies have measured airway remodeling and air trapping in patients with asthma using both qualitative, semi-quantitative, and quantitative CT/MRI techniques. Gupta and colleagues were the first to describe increased bronchial wall thickening in patients with severe asthma (n = 185) using CT  scanning and magnetic resonance imaging (MRI) has enabled the 3-dimensional (3D) assessment of the lung structure using CT . Later, using CT  are not using CT . Prior rusing CT . CT imagver time . This fiver time .Several studies have also tried to correlate measures of large airway wall remodeling on CT with histopathological changes within the airway wall. Aysola et al. demonstrated that airway wall thickness at the third generation, correlated with increased epithelial and reticular basement membrane thickness on histology . HoweverSeveral studies have used expiratory CT scans to analyze areas of low attenuation to identify areas of air trapping in asthmatic subjects . Air tra1/FVC), and blood eosinophils  which was also associated with increased sputum eosinophils and eosinophil peroxidase (EPO) levels  levels . Subsequ) levels . These d) levels . To eval) levels . This st) levels . Lastly,) levels . Togethein vivo in humans due to the small field of view and higher radiation dose. To date, research on asthma using microCT has been limited to murine models of asthma  , dependif asthma . These s1 and patients that undergo this procedure have an increased risk for bronchiectasis  aim to control symptoms and minimize the risk of exacerbations and persistent airflow limitation . As per iectasis . This coiectasis . Langtoniectasis . Thus, wIt was first shown in a double-blind randomized controlled trial, that 12 months of fluticasone propionate treatment decreases reticular basement membrane thickness, though there were no effects on collagen I and III deposition . ICS treWith regards to airway epithelial damage, Dorsheid et al. showed that treatment with dexamethasone, beclomethasone, budesonide, and triamcinolone on primary airway epithelial cells induced apoptosis . SeveralThere are limited studies on how ICS use affects the pulmonary vasculature in asthma. Notably, Hoshino et al. showed that 6 months of treatment with inhaled beclomethasone dipropionate lead to a significant reduction in vessel number and vascularity within the lamina propria . HoweverWhen assessing the effects of long-term use of ICS at high doses on airway remodeling, it is important to note the side effects of ICS which include impaired growth in children, decreased bone mineral density, and cataracts . As wellIn asthma, leukotriene receptor antagonists including zafirlukast and montelukast have been shown to decrease inflammation, improve airway hyperresponsiveness and reduce exacerbations . Most ofn = 24) results in a reduction of tenascin and lumican, two reticular basement membrane proteins that are upregulated in asthma. The mechanism in which mepolizumab may be modulating the ECM is not clear, though it has been proposed to be due to the reduction of eosinophils producing TGF-β1, reducing myofibroblasts and ECM protein expression  and chronic obstructive pulmonary disease (COPD) . Mucolyt2 antagonist in a phase 2 randomized, placebo-controlled trial, which reduced eosinophilic inflammation in the airways by blocking PGD2-driven release of Th2 cytokines and improved lung function , there was an 18% reduction of ASM thickness in severe asthma at the fourth generation, measured by CT, and additionally, that there was a significant decrease in ASM cell proliferation . The folfunction . Then, afunction . Though function . Finallyfunction . Though function .Thus, much further work is required to assess the responses of current pharmacological agents for asthma on airway remodeling. However, as it has been highlighted by earlier studies, the concept that inflammation and airway remodeling are tied may not be correct and targeted therapies focused on airway remodeling features may be required.in vivo imaging tools such as CT and MRI that are enabling measures of the airway wall in vivo and longitudinally in research studies. Importantly, such volumetric imaging tools provide the opportunity to assess the heterogeneity of airway remodeling within the whole lung and has led to the novel identification of heterogenous gas trapping and mucus plugging as important predictors of patient outcomes. However, still more work needs to be done to now assess how these radiological features can be understood pathologically and then translated to therapeutic targets. Lastly, we summarize the current knowledge of modification of airway remodeling with available asthma therapeutics to highlight the need for future studies that could now use in vivo imaging tools to assess airway remodeling outcomes with therapeutics.This review article provides an overview of the existing knowledge on airway remodeling features observed in asthma, including epithelial damage, mucus cell metaplasia, ECM remodeling in both the airways and vessels, angiogenesis, and increased smooth muscle mass. While such studies have provided extensive knowledge on different aspects of airway remodeling, they have relied on biopsy sampling or autopsy lungs which have limitations. Biopsy samples are limited in the airway wall structures that can be sampled and the tissue artefacts that can be induced when crunched by the biopsy forceps. Autopsy studies on lungs from fatal asthmatics offer a unique opportunity to assess the heterogeneity of airway remodeling within a lung, but it does not provide the opportunity to assess the spectrum of the asthma syndrome. In this review, we highlight the potential of