Atrial cardiomyopathy (ACM) represents a constantly evolving concept, with increasing importance in contemporary research and clinical practice. A better understanding of the mechanisms involved in atrial remodeling and its clinical correlations especially with atrial fibrillation (AF) and other cardiometabolic comorbidities may induce a significant impact on the diagnosis, prognosis, and therapeutic approach of ACM-related comorbidities. Although initially described several decades ago, investigators have only recently highlighted that several renal, metabolic, and cardiovascular diseases are determining factors for atrial remodeling and subsequent ACM. Based on data from multiple recent studies, our research emphasizes the correlations between ACM and other coexisting pathologies including cardiovascular, respiratory, or metabolic diseases, with fibrosis being the most incriminated pathophysiological mechanism. In addition to the usual tests, the paraclinical assessment of ACM is increasingly based on the use of various cardiac biomarkers, while the cardiac magnetic resonance (CMR) has become an increasingly tempting diagnostic too for describing morphofunctional aspects of the heart chambers, with the gadolinium contrast enhanced CMR (LGE-CMR) emerging as a commonly used technique aiming to identify and quantify the precise extent of atrial fibrosis. Further research should be conducted in order to clarify our knowledge regarding atrial remodeling and, therefore, to develop new and improved therapeutic approaches in these patients. Atrial cardiomyopathy (ACM) and its clinical importance were first described in 1972 by Nagle et al. [The aim of this review is to depict some coordinates of ACM, from the molecular level to the therapeutic approach, for a better understanding of the mechanisms involved in atrial remodeling. Additionally, we reviewed some particular ACM clinical correlations, especially with atrial fibrillation (AF) and the common AF-related thromboembolic events, that may have an important impact concerning the diagnosis, prognosis, and therapeutic decision-making process in ACM.High-incidence pathologies like chronic heart failure (CHF), AF, hypertension, diabetes mellitus (DM), and some other conditions including aging and obesity, are well-established causes of atrial remodeling. Although the induced changes share many similarities, there is an important histopathological substrate variability between the different clinical disorders ,3. ConseClass I: primarily cardiomyocyte changes ;Class II: predominantly fibrotic changes (cigarette smokers and the elderly);Class III: combined cardiomyocyte-pathology/fibrosis ;Class IV: primarily non-collagen infiltration, with or without cardiomyocyte changes  .There is an intimate relationship between atrial activity and ventricular systolic and diastolic performance; the main role of the left atrium (LA) is to manage the left ventricular filling pressures and cardiovascular performance by acting as a reservoir for the venous return during ventricular systole and as an additional pump for ventricular filling via an active atrial contraction ,4,5.Atrial remodeling is defined as a persistent change in the LA structure or function, possibly due to the appearance of atrial tachyarrhythmias such as AF and atrial flutter or secondary to pressure or volume overload related to CHF and other fibrotic disorders . FurtherAtrial remodeling is based on three principal pathophysiological pillars: structural, electrical, and functional remodeling.Atrial structural remodeling is characterized by increased interstitial fibrosis and by maladaptive structural changes secondary to inflammation, pressure, or volume overload, leading to atrial enlargement ,8. One oAngiotensin II and transforming growth factor beta-1 are major stimulators of collagen production and key regulators of fibrosis. Angiotensin II and aldosterone are involved in inflammation and oxidative stress initiation. Recent studies have shown that inflammation and excess production of reactive species of oxygen seem to play a principal role in atrial remodeling ,13. EvenAtrial functional remodeling is encountered in many pathological systemic processes and can cause alterations in atrial functioning regardless of the LA size ,9. This 2+) and potassium fluxes from the myocytes [2+ based on Ca2+ overload can affect cardiomyocyte excitation-coupling [Electrical remodeling consists of a complex electrical substrate and ionic mechanism changes in the atria . Factorsmyocytes . The inacoupling  promotincoupling . The moscoupling . Anothercoupling . Autonomcoupling .Over the past decades, investigators have shown that many cardiovascular diseases such as high blood pressure , valvulaHigh blood pressure represents one of the main cardiovascular risk factors for ACM . PreviouConditions highly associated with AF  are often complicated by increased hemodynamic atrial load, leading to the overstretching of the atrial myofibriles. Available in vivo and in vitro data suggest that significant strain leads to hypertrophy, dedifferentiation, and ECM remodeling, as well as to electrical alterations, all these pathways being associated with an increased risk of AF and further atrial remodeling, thus closing a vicious pathophysiological circle .Normal function of the LA is necessary for optimal ventricular filling and adequate overall cardiac performance. Atrial dysfunction due to changes in the ventricular structure is a common finding in patients with CHF ,14.The LA volume is a sensitive indicator of LV filling pressure and reflects the degree of diastolic dysfunction, in the absence of severe valvular disease and/or AF. In order to reflect the LA volume’s capacity to reveal the chronicity of diastolic dysfunction, it has been proposed as the “glycosylated hemoglobin of diastolic dysfunction.” Also, because of divergent effects of preload and mural compliance on transmitral velocities, the mitral inflow patterns may appear normal  despite abnormal filling pressures. In such situations, the LA volume index (LAVI) provides the highest discriminative value in distinguishing between normal and pseudonormal transmitral filling patterns .2 was an independent predictor of the first CHF episode [Why are these aspects important in patients with CHF? Mainly because the diastolic dysfunction represents the first phase of the so-called heart failure (HF) with preserved ejection fraction (EF > 50%). In these patients, a LA volume of ≥32 mL/m episode .A study conducted on 198 HF patients and 40 healthy controls compared the LA properties between HF with preserved and reduced ventricular ejection fraction. Investigators revealed that LA conduit, reservoir, and contraction functions are affected in diseased patients compared to control groups. Furthermore, they found that HF with preserved ejection fraction is associated with more dilatation and systolic dysfunction, while HF with reduced ejection fraction has an increased stiffness and susceptibility to AF .AF is the most frequent arrhythmia worldwide, affecting 25% of the adult population during their life . AF occuThe pathways leading to thrombogenesis in AF are various, and there is evidence to suggest that all three elements of Virchow’s triad are present in AF: abnormal stasis of blood flow, endothelial dysfunction, and hypercoagulability .The electrical and structural substrate within the LA in patients with paroxysmal and persistent AF has been studied by Teh et al. The investigators have demonstrated slower conduction, a higher proportion of decreased voltage, a lower global LA voltage, and an increased prevalence of complex signals in AF patients compared to the control group. These changes were more extensive in persistent AF .Cardiac fibrosis consists of excessive deposition of collagen fibers in the ECM. High collagen accumulation has been revealed in LA tissue obtained from atriotomy in cadaveric patients with AF . Stiles It is incompletely understood why some patients present repetitive episodes of paroxysmal AF for years or even for their entire lifetime, while other patients having identical comorbidities evolve into persistent AF in a rather short period of time. The above-mentioned concept of the negative “auto-amplification” that characterizes AF does not provide a detailed explanation for these differences. On the other hand, the results of atrial electroanatomic mapping in patients with AF and similar clinical findings were very different. These conclusions suggest the importance of a structural atrial condition defined as fibrotic atrial myopathy. However, even the presence of the most affected substrate can be shown in patients with paroxysmal AF instead of the persistent form ,29.An important link between AF and ACM is the inflammatory state . The levAF is a well-known risk factor for thromboembolic events such as ischemic stroke. In addition, atrial remodeling leads to a hypercoagulable state involved in thrombogenesis ,15. Spec-atrial hypocontractility persists for up to two months following AF conversion to sinus rhythm;-atrial dilation and hypocontractility may aggravate despite catheter ablation of AF ;-atrial hypocontractility alone (without concomitant AF) is an independent risk factor for stroke .Specific echocardiographic assessment of atrial wall velocities in AF patients revealed that there is a significant impairment of atrial contractility. Several hypotheses were raised concerning the link between atrial contractile dysfunction and the increased stroke risk:Both OSA and LA enlargement are conditions that are commonly found in patients with cardiovascular diseases. Severe OSA may also increase LA size, independently of other risk factors. Several pathophysiological mechanisms induced by OSA have been suggested to explain why this condition may trigger or aggravate ACM. Increased sympathetic activity and systemic inflammation due to intermittent hypoxemia, increased intrathoracic pressure during the apneas are all causing supplementary atrial wall stress .The basic mechanism by which OSA syndrome affects the atrial myocardial tissue is not completely understood. Dimitri et al.  investigDeleterious effects of hyperglycemia in DM go beyond alterations in metabolic homeostasis, also modifying the vascular endothelium and inducing both direct and indirect myocardial injury. Increased inflammation and oxidative stress induce the formation of advanced glycation end products (AGEs), cellular apoptosis, mitochondrial dysfunction, and disorders of myocardial metabolism. An essential physiopathological step is the capacity of AGEs to infiltrate the myocardium leading to interstitial fibrosis and hypertrophy. All these events, reunited into the term DM cardiomyopathy, determine the substrate for anatomic and electrical atrial remodeling .Additional negative prognosis factors are represented by an increasing prevalence of obesity, lack of regular physical activity, and exposure to stress, nowadays type 2 DM becoming one of the most common cardiovascular risk factors.Over time, many studies have evaluated the influence of LA structural and functional remodeling in patients with type 2 diabetes by using various imaging methods. In patients with DM, echocardiography findings reveal that LA dysfunction can be a consequence of LA enlargement . A recenAlthough many studies have shown that obese individuals have LA enlargement, given that the atrial size is typically quantified by indexing it to height or body surface, the final figures are relatively similar to non-obese controls. However, given the increased prevalence of obesity-associated comorbidities which can also influence cardiovascular remodeling, the real, independent impact of obesity on ACM can be difficult to assess .Epicardial fat is metabolically active and releases inflammatory cytokines, adipokines, and free fatty acids that are involved in the progression of atrial fibrotic remodeling. As a result, local epicardial adiposity can promote the substrate for arrhythmias. There is also a strong relationship between the quantity of pericardial fat and AF prognosis. Lifestyle changes, weight loss, normal blood pressure, and optimal glycemic values were connected with a better rhythm control among obese patients with AF after catheter ablation .2) was related to adverse cardiovascular events and all-cause mortality [The prevalence of AF increases with age in line with a decline in the estimated glomerular filtration rate (eGFR). Various reports claim that 7–18% of patients with CKD had AF, while 10–33% of patients were incidentally found with CKD at the time of the newly diagnosed AF. The occurrence of AF may promote the progression of CKD and increase the risk of developing end-stage renal disease. Moreover, the coexistence of AF and CKD . A proposed algorithm is found in According to current literature data, the dosing of novel biomarkers, as well as imaging explorations aimed to quantify and identify fibrosis, can be effective for the diagnosis of atrial remodeling.Multiple studies have demonstrated that high blood levels of CRP and IL-6 are associated with an increased incidence of AF, both among patients undergoing cardiac surgery and the general population. Increased levels of the above-mentioned inflammatory biomarkers are also correlated with a significant risk of arrhythmia recurrence in patients who underwent electrical cardioversion or catheter ablation. Further supporting these findings, a recent study showed a decline in CRP levels after the conversion to sinus rhythm. Very interesting, the duration of AF does not influence the level of these markers ,13.IL-6 can also interfere with the pro-thrombotic state during AF, by endothelial activation and platelet aggregation ,10. IncrAn increased level of N-terminal pro-B-type natriuretic peptide (NT-proBNP) represents an established hallmark in patients with HF, however, this biomarker is markedly influenced by the presence of concomitant AF, making it difficult to distinguish between HF versus AF as the primary determinant of a raised NT-proBNP. To shed some light on this controversy, a very recent study compared the plasma levels of NT-proBNP in patients with HF in AF versus HF in sinus rhythm and found that baseline NT-proBNP serum concentrations of patients who had AF were higher than those of patients in sinus rhythm, regardless of whether the latter had a history of AF or not . MoreoveAtrial changes induced by AF, such as increased atrial size and fibrosis, stimulates the secretion of natriuretic peptides. Atrial natriuretic peptide (ANP) is a cardiac hormone with vasodilatory, antihypertrophic, diuretic, and natriuretic effects. Therefore, ANP is involved in maintaining the water-sodium balance and optimal blood pressure. A previous study revealed that mice with ANP deficiency developed hypertension and cardiac hypertrophy as a result of ischemic remodeling and pressure overload. More recently, ANP has been shown to have pro-angiogenetic, anti-inflammatory, and anti-atherosclerotic properties, being consequently involved in the underlying mechanisms of cardiac remodeling ,43.Given the increased prevalence of AF and atrial remodeling among patients with HF, several studies aimed to evaluate the utility of HF biomarkers in detecting ACM. Denysiak et al. proved that soluble interleukin 1 receptor-like 1 (ST2) is a marker of advanced functional remodeling in patients with AF, and is associated with significantly longer ablation procedures independent of LA size , while oAnother promising biomarker of cardiac fibrosis is represented by syndecan-1, especially in patients with HF with preserved ejection fraction (EF). Given the predominance of diastolic dysfunction in these patients , an early assessment of the myocardial fibrotic injury is paramount in determining the chances for cardioversion or, if not the case, of maintaining an adequate heart rate .Numerous imaging explorations are used currently in clinical practice to identify LA remodeling . EchocarSome new techniques, like the evaluation of the LA volume by 3D-Echocardiography, represent more accurate methods than the classic determination of the LA size. Two-dimensional speckle-tracking echo is a more sensitive imaging method for the early assessment of functional remodeling, even before the appearance of visible anatomical changes in biopsies . Global Cardiac computed tomography is superior to 2D-Echocardiography in the assessment of atrial volumes, and it can be used in thrombus screening before undergoing AF ablation ,47.p < 0.001), even after adjusting it to LA volume. Moreover, combined evaluation of LA EAT and LA volume provided additive diagnostic value and superior performance in the detection of AF compared to the assessment of online LA volume (p < 0.001) [In the light of some latest research, the assessment of epicardial adipose tissue (EAT) depots in cardiovascular pathology is of particular interest. Various studies that successfully used CMR for the evaluation of EAT volume and thickness found interesting correlations between the presence of fatty tissue and the atrial remodeling, highlighting areas of low voltage, conduction impairment, and greater fractionation of electrograms in anatomical regions adjacent to significant epicardial fat depots . A study< 0.001) . Very in< 0.001) .Even if CMR is considered the gold standard for visceral adipose tissue assessment, the majority of clinical studies that assessed EAT to date were performed using computed tomography (CT), because of its adequate spatial resolution, excellent reproducibility, and ease to perform. Additionally, in several situations, EAT was quantified on a CT scan performed for other pathologies . The resA novel noninvasive method used for the assessment of atrial flow dynamics is the 4-dimensional (4D) flow CMR. This method is more accurate than the trans-esophageal echocardiography and provides a comprehensive detection and quantification of stasis in the LA and LA appendage (the most prone location for thrombus formation) .Electroanatomic mapping is the standard invasive method in ACM substrate characterization, through the geographic display of signal amplitude data and by rendering the atrial surfaces. It can be used to investigate atrial myopathy associated with sinus node disease, rheumatic mitral stenosis, atrial septal defects, CHF, OSA syndrome, and aging .Cardiac remodeling includes functional, electrical, and structural changes that are closely interconnected, and influence both the therapeutic approach and the prognosis. The practical management of ACM is based on stroke prevention, cardiac rhythm therapy, rate control, and ablation strategy .Amiodarone is the most effective and frequently used antiarrhythmic drug. However, recent recommendations emphasize catheter ablation rather than amiodarone for AF treatment . PreviouThe importance of metabolic effects on ACM is supported by the fact that weight loss and risk factor management induce better post-ablation results and a decreased rate of AF recurrence ,24. HoweFurthermore, other therapies have been shown to be useful in atrial remodeling. ANP promotes natriuresis, diuresis, vasodilation, and inhibits aldosterone and renin secretion. Given its effects, ANP is considered a promising drug for various cardiovascular diseases. It has been proven that ANP administration reduces maladaptive cardiac remodeling and has anti-inflammatory and proangiogenic effects. The inhibition of neprilysin results in an increased ANP secretion. Therefore, the inhibition of neprilysin and angiotensin receptors is a novel therapy in patients with HF . Thiazol2+- sensitizing agent levosimendan. This option is sustained by some evidence concerning dysregulation of the myofilament response to Ca2+ release or Ca2+ impaired sensitivity, which are significant contributors to contractile dysfunction in AF [2+ homeostasis and subsequent contractility efficiency rely upon phosphorylation processes, which, in AF, are pathologically altered. Some recent studies have shown that levosimendan increases cerebral blood flow, decreases NT-proBNP , and improves atrial pump function on echocardiography; however, it also increases the occurrence of AF in these patients, an undesired effect attenuated by concomitant treatment with ranolazine, an inhibitor of the late inward sodium current [Another potential therapeutic target in improving atrial contractility is the Caon in AF . Intrace current .Some very recent reports were focused on unravelling the pleiotropic, cardiovascular protective effects of sodium–glucose cotransporter-2 (SGLT2) inhibitors, a novel class of oral hypoglycemic medication that showed very promising results in the treatment of HF. A study conducted by Shao et al. revealed that empaglifozin, a SGLT2 inhibitor, can prevent atrial structural and electrical remodeling as well as ameliorate mitochondrial metabolism in diabetic rats, via the peroxisome proliferator-activated receptor-c coactivator 1α (PGC-1α)/nuclear respiratory factor-1 (NRF-1)/mitochondrial transcription factor A (Tfam) signaling pathway . MoreovePrevious studies have drawn an interesting new direction concerning the possible roles of oxidative stress and inflammation in the mechanisms of the electrical and structural substrates for atrial remodeling and subsequently, AF ,58. EvenIt was demonstrated that another SGLT2 inhibitor, canaglifozin, exhibited significant antioxidant properties in the atrial tissue. This effect was related to increased ketogenesis promoted by canaglifozin, thus enhancing the myocardial uptake of ketoacids , which competes with fatty acid oxidation. Cardioprotection is based exactly on this metabolic switch, the important metabolic efficiency of ketone body oxidation being well-established in the literature . Given tCardiac resynchronization therapy (CRT) has been shown to improve not only the LV ejection fraction but also LA function. A study including a total of 41 patients who received a de novo CRT implantation revealed that the P-wave duration measured in the surface 12-lead ECG significantly decreased following CRT therapy (at least 12 months since implantation and with adequate bi-ventricular pacing > 92% of the time). This is an important clue regarding the therapeutic potential use of CRT in patients with ACM because the prolongation of the P-wave duration represents a quantitative indirect marker for atrial electrical remodeling .CRT’s capacity to induce LV and LA reverse remodeling might be partly related to the improved LV filling and subsequent increase in LV contractility, cardiac output, and LV ejection fraction. In a similar manner, ameliorated diastolic function and optimal LA filling are also enhancing LV contractility. Furthermore, synchronous triggering of ventricular depolarization also decreases mitral regurgitation, thus contributing to LA reverse remodeling ,63,64.The results from a MADIT-CRT  study comprising 533 patients with CRT and defibrillator (CRT-D) showed that >40% of the patients presented complete left-sided reverse remodeling  and their increasing availability in patients with end-stage HF play an important role in the prognosis of patients with impaired atrial function. Commonly used either as a bridge therapy to heart transplantation or following precise, limited indications, LVADs can be an arrhythmogenic trigger, as was shown in many studies that highlighted the frequency of AF occurrence in both pre-LVAD or post-LVAD patients with advanced HF, with an estimated prevalence of 21–54% and a great impact upon patients’ outcome and mortality rates ,66. MoreUnder these circumstances, it was hypothesized that LVAD implantation may have a significant impact upon atrial remodeling through modifications in atrial filling pressures and myocardial strain. A recent study that evaluated several echocardiographic atrial parameters before and after LVAD implantation pointed out that LA size and volume index were significantly decreased after LVAD implantation, and a significant proportion of patients with previous paroxysmal AF presented no further documented AF episodes. Nevertheless, the occurrence of a post-implantation, new-onset AF was associated with poor outcome and increased mortality in patients receiving LVAD. Basically, these latest insights suggest that LVAD induces reverse atrial electrical and structural remodeling, but arrhythmic events may still severely influence the clinical outcome of patients with LVAD .p < 0.001) and six months  were significantly smaller compared with just after ablation in the patients without AF recurrence. Additionally, the authors found a significant decrease of the natriuretic peptide concentrations between baseline levels and those at three and six months , thus furtherly confirming the potential benefits of catheter ablation not just for AF management, but also for the outcome of patients with ACM and HF [Even if the topic is too vast to be exhaustively covered by this review, we need to mention the anti-remodeling role of catheter ablation for isolated AF which was confirmed in multiple studies during the last two decades ,69,70. TM and HF .In recent years, novel computational models and applications for atrial remodeling entered the spotlight, thus offering a framework for a better approach to AF ablation, pharmacotherapy, and the subsequent long-term prognosis of these patients .Basically, these complex, multi-scale atrial models are mathematical models that are bridging the molecular, cell-scale mechanisms with the tissue alteration and finally, with the whole atria scale dysfunction, integrating this pathophysiological continuum into a graphical representation of atrial electrophysiology .The cell scale provides information concerning the kinetics of different ionic channels and the coupling of some regulatory proteins in order to produce the transmembrane potential of the atrial myocytes, while the tissue scale highlights the intercellular coupling and fiber orientation that exerts a great influence upon electrical propagation. Finally, the whole atria scale of these computational models provides insights into the segmentary distribution of normal tissue or fibrotic areas, also generating a 3D reconstruction of the atrial geometry that simulates atrial activation .+ channel blockade with repolarization modulating drugs to generate ideal compound profiles that could reverse atrial remodeling or at least inhibit its underlying electro-structural mechanisms [The clinical utility of computational relies on their capacity to allow the study of some drugs’ effects on a wide range of cellular phenotypes, thus providing an improved prognosis tool concerning the impact of certain substances on the ionic currents. For example, recent research emphasizes the development of multi-target therapies that aim to combine the efficacy of Nachanisms .Another important aspect with direct clinical impact refers to the pharmacokinetic (PK) and pharmacodynamic (PD) modeling of some antiarrhythmic substances such as drug conversion from amiodarone to its derivative dronedarone. Both substances exhibit a similar main structure (removed iodine and added methanesulfonyl group) and overall electropharmacologic profile but with different effects on individual ion channels. Moreover, concerning PK models, amiodarone has increased affinity to accumulate in different tissues due to a longer half-life, thus raising the risk for non-cardiac side-effects. In this context, PK and PD modeling should be routinely considered for drug development, providing a better understanding of several dynamic drug–ion channel interactions involved in ACM and their subsequent positive or negative (non)cardiac outcomes .ACM represents a complex entity that is correlated with numerous commonly met conditions from clinical practice, including systemic hypertension, congestive HF, AF, type 2 diabetes, sleep apnea syndrome, and obesity. Fibrosis is the most important pathophysiological mechanism of atrial remodeling, its accurate assessment representing the foundation for an adequate therapeutic approach and an improved long-term prognosis.Besides the classic inflammatory biomarkers or natriuretic peptides, laboratory diagnosis of ACM is focused on modern biomarkers that could indicate the magnitude of the fibrogenic process and also assess the risk of future cardiovascular events. CMR has become a promising and increasingly used technique for describing heart chamber structure and function, LGE-CMR being commonly used to assess the extent of atrial fibrosis and offering an accurate characterization of scar tissue formation after the ablation procedure and thus ascertaining the need for an eventual reintervention.The ultimate goal will be to translate recent molecular and clinical findings into an algorithm-based therapy for patients diagnosed with conditions suggestive of ACM. In addition to classical pharmacological drugs used in HF or AF, invasive devices  or the use of computational models may be a viable option for both the acute and long-term therapeutic optimization of patients with HF and ACM.


These include, among others, connective tissue, cartilage and bone of the face and skull, neurons and glia of the peripheral nervous system (including enteric nervous system), and melanocytes. Following migration, these derivatives extensively populate multiple germ layers. Within the competent neural border ectoderm, an area located at the junction between the neural and non-neural ectoderm during embryonic development, neural crest cells form in response to a series of inductive secreted cues including BMP, Wnt, and FGF signals. As cells become progressively specified, they express transcriptional modules conducive with their stage of fate determination or cell state. Those sequential states include the neural border state, the premigratory neural crest state, the epithelium-to-mesenchyme transitional state, and the migratory state to end with post-migratory and differentiation states. However, despite the extensive knowledge accumulated over 150 years of neural crest biology, many key questions remain open, in particular the timing of neural crest lineage determination, the control of potency during early developmental stages, and the lineage relationships between different subpopulations of neural crest cells. In this review, we discuss the recent advances in understanding early neural crest formation using cutting-edge high-throughput single cell sequencing approaches. We will discuss how this new transcriptomic data, from 2017 to 2021, has advanced our knowledge of the steps in neural crest cell lineage commitment and specification, the mechanisms driving multipotency, and diversification. We will then discuss the questions that remain to be resolved and how these approaches may continue to unveil the biology of these fascinating cells.The neural crest is a unique population of multipotent cells forming in vertebrate embryos. Their vast cell fate potential enables the generation of a diverse array of differentiated cell types  Alternatively, neural crest cells are selected using a reporter gene and further processed . Spatial organisation is lost during the process.Whole embryos  is identified by a unique barcode for later demultiplexing.3- Sequencing and associated quality controlsPooled cDNAs are sequenced using next-generation massively parallel sequencing. Sequences are filtered by several quality control steps, aligned on the reference genome for each cell, and a count matrix is generated. Cells with sufficient count numbers are selected for further analysis.4- Clusteringcollectively cover the transcriptome of the population. Each cluster is identified using known markers.The depth of sequencing using one cell is very limited. Most expressed genes are not captured. Compared to bulk sequencing (reading over 90% of the transcriptome), single cell transcriptomes barely cover 10% of the expressed transcripts; for this reason, similar cells are clustered back and https://hbctraining.github.io/In-depth-NGS-Data-Analysis-Course/sessionIV/lessons/SC_pre- QC.html5- Differential gene expression analysis and trajectory analysis are conducted at the level of clusters using a variety of computational methods. For example, check in vivo by its anatomical position, by the selective expression of marker genes/proteins, or after labelling with a lineage tracer. Alternatively, a cell population can be isolated by microdissection or fluorescence-activated cell sorting (FACS) to be studied in vitro or in vivo. In contrast, previous transcriptomic and epigenomic analyses have mostly been conducted on whole embryos or large cell populations by "bulk" sequencing of the RNA expression profile of a population: examples include developmental series of whole embryos in Xenopus, zebrafish, and mouse4. More recently, transcriptomes and epigenomes of selected embryonic tissues were isolated by either manual or laser-mediated dissection or using FACS to separate labelled cell groups. These datasets were analysed with or without subsequent spatial reconstruction. Examples include the early mouse embryo5, developing blastula-stage half embryos6, the gastrula-stage mesoderm7, the gastrula and neurula-stage dorsal ectoderm in Xenopus laevis embryos8, the dorsal neural tube/neural crest in lamprey embryos9, or foxd3-positive neural crest cells in chick and zebrafish embryos11. Also using zebrafish, additional bar-coding and temporally controlled gene editing strategies . We particularly focus on the two key questions addressed in these studies: the rules of neural crest lineage diversification and the mechanisms driving its multipotency.The neural crest is an early multipotent cell population that emigrates from the edge of the closing neural tube during neurulation and organogenesis stages and populates each germ layer of the vertebrate embryo body either directly during early embryogenesis or by the dissemination of derivatives later on . DiscoveLineage tracing is particularly difficult to complete for the neural crest because of the extensive dispersion of its derivatives across all tissues and organs: rare neural crest-derived cells continue being discovered after 150 years of fate mapping . A remaining yet essential gap in our knowledge is the exact pattern of fate decisions in vivo resulting in the stereotyped formation of each lineage. Correlated to this essential question, the molecular mechanisms endowing the neural crest with its extraordinary multipotency remain poorly understood, and the few models proposed so far remain open to debate. In this context, a detailed series of single cell transcriptomic and genomic data could inform the existence of neural crest progenitors with progressively more restricted fates.Importantly, neural crest cells contribute to more than 30 different cell types, either directly during early stages of development or by a later contribution of neural crest-derived multipotent progenitors located along the nerves , during neural crest epithelium-to-mesenchymal transition (EMT) at the end of neurulation , during neural crest migration, and finally during its differentiation during organogenesis  regulators, please refer to 21, DNMT3a/b22, and PRDM proteins24), cell–cell or cell–matrix adhesion molecules and migration regulators (SDF-1 ligand and its receptor Cxcr426 and N-cadherin27), and novel regulators of signalling pathways . Importantly, the NC-GRN is largely conserved throughout vertebrate evolution. As the palette of animal models used in neural crest studies grows, including earlier- and later-derived vertebrates, the logic of evolutionary modulations becomes available on top of the conserved backbone of the NC-GRN34.In the last 15 years, a cohort of gain- and loss-of-function studies has set up the scaffold of a complex yet still incomplete network of epistasis regulations between key transcription factors controlling neural crest specification in the gastrula neural/non-neural border ectoderm  and melanocytes (pigmented cells) differentiation is also compared.@Human differentiation markers used are neuronal.41. It should be noted that this strategy of inferring a cell's fate using its gene signature at one time point contrasts with the more classical approaches that relied on the actual differentiation of several distinct cell types from a living single cell and thus did not "infer" cell identity but rather cell fate was demonstrated from end-point analysis44. Furthermore, single cell approaches unavoidably lose the spatial/anatomical information and define groups of cells (clusters) by their expression of known gene markers; this strategy heavily relies on the accuracy of the developmental markers used as a diagnostic of each lineage. In sum, expectations from neural crest-related single cell genomics are high but the limitations can readily be anticipated.In the context of the well-advanced NC-GRN, scRNA-seq is expected to confirm the identity of existing neural crest subpopulations, to inform previously unidentified novel groups of neural crest cells, and to provide extended gene signatures for each group of cells. Moreover, single cell genomics will provide evidence for neural crest successive states of development  using trXenopus, chick, and mouse. Although most of these studies were not designed specifically to study neural crest biology, they provide important insights. After describing a few general features taken collectively from the datasets, we discuss three main points using the model organism best suited to address each of them and then compare the results between species  and 8 stages between sphere and prim5 (4–24 hpf)45 respectively. These studies support previous results that suggest that cell fate plasticity and canalisation during differentiation happens in a progressive manner  but also observed in Farrell et al., where progenitors can give rise to multiple cell fates of endoderm and non-chordal mesoderm . In X. tropicalis, a large series of 8 successive developmental stages from blastula (stage 8) to early tailbud (stage 22) was investigated by scRNA-seq3. Data from this sequencing showed that developmental states do undergo tree-like state transitions, similar to binary decisions but with more possible branches similar to other lineage trees, and that most of the gene expression modules are expressed earlier than previously recognised.The first use of scRNA-seq in zebrafish and e manner . Each po45. In Xenopus, however, the first step from the blastula state is the formation of the three embryonic germ layers  along with the separate (pluripotent) germ line at the early gastrula stage3 . When single cell transcriptomics is combined with fate mapping of wild-type and mutant embryos, these data suggest that not all zebrafish cell fates have tree-like cell fate trajectories but undergo defined steps of molecular specification4 . The final overall conclusion from these studies is that mutant cells  do not adopt new cell fates on the transcriptional level but express a transcriptional profile similar to that of wild-type cells, suggesting canalisation or a cell fate that is similar to that which wild-type cells would normally differentiate into , and this is at around the same or at an earlier time point than the separation between different germ layersication4 . While tate into .in vivo, branch away separately45 4. Transcriptionally, tailbud-stage (specified) neural crest cells would be more similar to posterior central nervous system than to neural border cells. In contrast, in Xenopus, Briggs and colleagues found that the neural crest lineage branches out early on from the neurectoderm state, itself directly linked to ectoderm3. The Xenopus data thus detect an earlier transcriptome diversification between neural and neural crest fates than in zebrafish. It would be interesting to directly compare the general neural crest specifier genes and the antero-posterior axis markers that influence data clustering in each study.In the zebrafish ectoderm lineage tree, inferred from scRNA-seq trajectories, the neural crest lineage branches close to the spinal cord trajectory, while the neural plate border cells, from which neural crest cells arise X. laevis, the earliest snail2 expression is detected at stage 11.5–12 by in situ hybridisation and whole embryo bulk RNA sequencing46, while neural border specification occurs during early gastrulation (stage 10.25)50. However, recent work has suggested that neural crest specification GRN is set up even earlier at early gastrulation or blastula stages51. The two latter studies explanted early stage blastula cells and showed that the cells were able to differentiate into neural crest cells autonomously when placed ex vivo. Sequencing of FAC-sorted neural crest cells has also been performed in zebrafish and mouse11. It is now possible to test the idea of the timing of neural crest specification at the level of gene expression . It seems in Xenopus and zebrafish that the specification occurs at mid- to late-gastrulation stages during normal development . Moreover, this idea was supported by neural crest cell sorting prior to sequencing using regulatory elements driving expression of the neural crest specifier foxd3 in zebrafish at 4 stages . Interestingly, sorting and sequencing in foxd3 mutants suggests that the GRN is very interactive, with foxd3 both activating the GRN of neural crest and repressing other genes in a likely mutual regulatory circuit11. Together, these data suggest that by selected gene expression analysis, the activation of the current NC-GRN occurs at the time of gastrulation rather than at the blastula stage. However, it would be important to further identify the gene regulatory circuits underlying the observations of early determination in the chick embryo blastula to compare to data from other species.Textbook descriptions of neural crest development suggest that specification occurs at the border between neural and non-neural ectoderm at the end of gastrulation or beginning of neurulation. In 55, while other studies have shown that some populations are more restricted59. In vitro, the clonal analysis of single neural crest cells cultured in the presence of various cytokines has provided a fate lineage tree of the chick cranial neural crest, demonstrating multiple degrees of potency, from progenitors restricted to a unique fate to the multipotent cells giving rise to all known neural crest derivatives  and endoderm (endodermin), demonstrating that neural crest cells can express markers of the three germ layers and are thus truly pluripotent. From these similarities, this study proposed that neural crest progenitors retain a genetic signature controlling pluripotency, suggesting a model in which a subset of cells at the neural border remain similar to blastula cells and pluripotent, while the rest of the germ layers become more restricted and, in the case of dorsal ectoderm, adopt a neuroectodermal fate61. This model contrasts with the classic germ layer model  of pluripotency/pleistopotency/multipotency and the ability to follow the developmental lineage of the multipotent progenitors and highlighting the evolutionary appearance of multipotent neural crest cells.A major outcome of single cell genomics would be to highlight the molecular basis of neural crest cells’ exceptional multipotency  strongly express Oct4 and, at lower levels, Nanog at the 4-somite stage (E8.0). They showed that in vivo the early neural folds do not express Oct4 (stage E7.75) but that Oct4 expression is activated in the neural folds at a later stage. By genetic labelling and genetic ablation studies, they further demonstrated that Oct4-positive cells generate craniofacial neural crest cells with an ectomesenchyme bias: Oct4 depletion affects ectomesenchyme derivatives but not neuro-glial fates, similar to mechanisms observed for nanog/ventx2 activity in other species68.Moreover, recent data from mouse cranial neural crest cellsXenopus and mouse is strong evidence that neural crest cells transition through neuroectoderm fate at the time of specification and re-activate a pluripotency circuit involving factors such as Oct4/PouV and Nanog/Ventx. Additional supporting preliminary data from mouse suggest that some genes, but not all of the pluripotent network, function in neural crest cells . Altogether, data from the new single cell sequencing approach more likely support the classical neurectoderm hypothesis , i.e. hatching gland cells that are unique to fishes and frogs. In addition, the specific lineages identified have multiple shared intermediate states. For example, both species have a population of progenitors that give rise to neural crest-derived xantophores, but they differentiate at different time points in development. Elegant single cell sequencing analysis of post embryonic and adult zebrafish pigment cells suggests that progenitors remain in adults and that thyroid hormone promotes xantophore and melanocyte maturation but in different ways76. Moreover, unique cell populations were also identified for each species: for example, the specialized epidermal cells arising in Xenopus embryos. Perhaps not surprisingly, there is a conserved reuse of transcription factor modules during development. Some of these factors, including foxd3 in the neural crest, are expressed within a lineage to define cell fate as a master regulator. Foxd3 both activates and represses gene expression within this lineage and thus has “bimodal” activity in the regulation of neural crest specification and later differentiation10. Similarly, in chick and mouse, Foxd3 regulates multipotency during specification and later promotes specific fates  and inhibits others (pigment)81. However, Foxd3 and other regulators are often expressed in more than one cell lineage, suggesting the importance of combinatorial reuse of these factors. In terms of evolution, scRNA-seq may also be able to give us clues about the relationships between cells in different organisms. Recent work performed in the ascidian Ciona intestinalis examined the expression of genes including Galanin in bipolar neurons that is also expressed in a neural crest derivative, dorsal root ganglion neurons, supporting previous work showing that these urochordate neurons arise from the neural plate border and migrate in a similar manner to neural crest cells83. Together these studies of whole embryo and neural crest-selected scRNA-seq give us clues to how overall cell fate is specified informing neural crest cell specification in general and how they arose during evolution.Interestingly, when the whole embryo 84. Second, for the most part, the sequencing by this method is very shallow, meaning that this approach resolves only the most highly expressed transcripts within neural crest cells from which we are proposing broad and general conclusions. This limited sequencing depth prevents evaluation of the low-level activation of gene expression, which is a critical factor for the understanding of the branch points of the NC-GRN. Additional approaches using nuclear nascent RNA sequencing or transcription at the enhancer sites may nicely complement scRNA-seq and better highlight the subtle dynamics of gene activation72. And, third, scRNA-seq has not yet been used to inform how the signalling response is integrated with targeted gene expression in individual cells or by nature and does not resolve important environmental cues that are required for neural crest specification. Specific to neural crest development, future work should 1) increase sampling time points for scRNA-seq to closely capture the intermediate states of the highly dynamic NC-GRN, 2) explore if cell death and elimination between two states influences the neural crest lineage tree by analysis of the apoptotic cascade, and 3) expand the scRNA-seq analysis from neurulation to differentiation time points and include more derivatives during neural crest development. Along these lines, the recent exploration of mouse cranial-most neural crest, using 4 successive stages between 4- and 10-somite stage, has provided additional insights into premigratory and migratory neural crest patterning and pluripotency68. All together, we believe that the future is bright. With improvement of the depth of sequencing, integration with spatial expression, and novel bioinformatic approaches, scRNAseq will continue to greatly enhance our understanding of neural crest biology.In this review, we have addressed how the innovative single cell approaches available so far inform and add to the knowledge base of neural crest cell specification, multipotency, and diversification. Reassuringly, the single cell data confirm the current understanding of the NC-GRN. However, the thorough analysis of migration and differentiation steps remains to be explored, as well as the analysis of the earliest timepoints of neural crest specification, despite a few important studies on specific neural crest subpopulations. Importantly, these new approaches allow for a comparison of transcriptomes across species and hopefully will resolve long-standing questions regarding neural crest cell specification, multipotency vs. pluripotency, and diversification. Current scRNA-seq along with other experimental studies support the classic hypothesis that neural crest cells arise from neuroectoderm and regain multipotency rather than maintaining a blastula-type pluripotency. However, there are still outstanding questions and technical limitations linked to the completed analyses. For one, scRNA-seq cannot resolve spatial relationships between cell populations. These data would have to be combined with more classical lineage tracing and fate mapping or by new exciting single cell approaches that map sequencing back to spatial expression


Currently, the significance of fungi as human pathogens is not medically concealed in the world. Consequently, suitable recognition and treatment of such infections are of great importance and necessitate the need for comprehensive information in this regard. The introduction of new antifungals and their use today, especially in the last two decades, have revolutionized the treatment of fungal infections. On the other hand, increasing drug resistance in the world has overshadowed such developments. The use of NPs results in the treatment of fungal infections and owing to their specific properties, these particles, unlike the pure antibiotics, can exert a greater inhibitory power although with less concentration compared with conventional drugs. Important reasons that have led to the use of antifungal drugs in delivery systems include reduced drug efficacy, limited penetration through tissue, poor aqueous solubility, decreased bioavailability, and poor drug pharmacokinetics. It is therefore hoped that unfavorable properties of antifungal drugs be mitigated via their incorporation into different types of NPs. This review summarizes the different types of NPs as delivery systems of antifungal as well as their advantages over pure drugs.  Nowadays, the significance of fungi as human pathogens is medically transparent in the world. Fungi that were not previously infectious in humans are nowadays among the opportunistic pathogens which are increasing in number. Thanks to their ability to adapt to a variety of environmental conditions, these fungi can easily endanger the lives of immune-deficient patients and are now one of the leading causes of death for these patients. Therefore, timely recognition and treatment of such infections are of great importance and necessitate the need for comprehensive information in this regard. Studies show that more than 300 million people suffer from serious fungal infections, accounting for about 1.4 million deaths annually . The inToday, nanoparticles (NPs) have been highly appreciated for their wide range of applications to various biological, pharmacy and medical fields. Structurally considered, their size barely exceeds the range of 100 nm. A broad spectrum of drugs, such as small hydrophobic and hydrophilic drugs, vaccines and biological molecules can be controlled by these NPs (Gupta and Xie 2018). NPs arNPs can be categorized in a variety of ways in terms of their size, shape and constituting materials Figure 1. Even thChitosan, a carrier of NPs, is a plentiful natural polysaccharide known for its beneficial properties. Compared to the synthetic polymers, chitosan obtained by chitin acetylation is suitable for the formation of nanogels due to its biocompatibility, better biodegradability, and stability as well as low toxicity . The usin vivo investigations indicated that the accumulation of drug-loaded nanoparticles in lungs was significantly higher than that of a pure drug; in addition to this, the prolonged retention of particles in lungs was observed . Sometial., 2020. So in gal., 2020). The beal., 2020). In thiRecent clinical and pre-clinical studies have revealed that targeted drug delivery systems are a great way to treat various life-threatening diseases. Smart drug delivery is divided into the following categories: Drug delivery using nanocarriers is absolutely desirable due to their very small diameters (10 to 1000 nm) which greatly contributes to improved encapsulated drug treatment efficacy . In recNanoliposomes are self-forming nanostructures that come together in an aqueous solution of lipid molecules. Phospholipid lipophilic molecules are grouped in such a way that their hydrophobic groups are directed inward whereas their hydrophilic groups are aligned in the outer layer of the sphere, thus forming a spherical bilayer membrane. This mode of orientation allows the loading of hydrophilic drugs in the nucleus, and hydrophobic agents in the liposomes shell Figure 2 . In thiSolid lipid NPs (SLN) are colloidal structures that can be prepared by emulsification and by means of mechanical forces such as ultrasound and homogenizer, they get reduced to below micrometers in size. These systems are formed by replacing the w/o emulsion oil phase with a solid oil or a mixture of solid oils; that is, a mixture of lipid matrix particles that are solid at room temperature and body. SLN consist of 0.1 to 30 % solid fat dispersed in the liquid phase and if necessary, 0.5 to 5 percent of the surfactant is also used in their preparation. The average SLN particles range from 40 to 1000 nm. Studies have shown that the physicochemical and stability properties of drugs loaded in the SLN depend on the properties of the drugs and the components used. These structures are capable of carrying drugs and active substances in their lipid portion, thereby protecting the target material from environmental damages. As a result, this spectrum of NPs can be used to deliver drugs and prolong their effectiveness . UnlikeNanocapsules consist of a thin membrane surrounding a core  with their size ranging from 10 nm to 1000 nm. Nanocapsules are submicroscopic colloidal drug carrier systems composed of an oily or an aqueous core surrounded by a thin polymer membrane. The membrane may be composed of natural or synthetic polymers . Two tePro-drugs are inactive biological compounds that become active after encountering a specific physiological barrier . The prThe main classes of antifungal medications used in mycoses treatment include polyenes , hamycin, natamycin, nystatin), azoles , triazoles , and thiazole (abafungin)), pyrimidine analogue (flucytosine), echinocandins , squalene monooxygenase , benzylamine (butenafine)), mitotic inhibitor (griseofulvin)  is a common cause of morbidity affecting millions of women worldwide; however, it is hoped that by further studies on this new formulation and clinical trials, this drug can be used to treat the so-called patients . Anotheal., 2016). Cassanal., 2016) investial., 2016). Anotheal., 2016) worked al., 2007). A studal., 2007). In 201al., 2010). Also, sny, 2016). One ofal., 2015). Other al., 2015; Al-Badral., 2015). One ofal., 2015) that conha, 2016). Anotheal., 2017). Anotheal., 2017). EL-Houal., 2017) generatal., 2017). All ofAnother SLN study was carried out on GF, a drug frequently used to treat dermatophytosis. However, today, considering the several shortcomings of this drug such as its poor oral bioavailability and some other side effects, the topical application of drug delivery system of GF has been proposed. For this purpose, Anurak and his colleagues in 2011 developed a GF-loaded SLN by microemulsion method. The researchers showed that using this formulation makes GF release profiles be a delayed-release and also makes large scale production of SLNs quite viable .Another drug delivery system is nanostructured lipid carriers (NLCs), being composed of both solid and liquid lipids as a core matrix . The utin vivo study and reported that using these NPs could facilitate the oral delivery of AmB .Cubosomes are extremely firm NPs formed from the lipid cubic phase and stabilized by a polymer-based outer corona . The cuCandida albicans and Candida tropicalis using AmB-conjugated biogenic silver NPs (AmB-bAgNPs). What they found in this study was that AmB-bAgNPs have a significant effect on enhancing their antifungal activities against Candida spp. compared to pure AmB. It was also reported that the antifungal stability of AmB-bAgNPs remained higher than the pure AmB for several days and this formulation could reduce the therapeutic cost and dose of AmB as well as its toxicity , the researchers reported that the formulation had a higher antifungal trait. They, therefore, suggested that using these NPs could open up a new avenue in the treatment of pathogenic fungi responsible for severe mycotic infections  manifest both biofilm inhibition capacity for Candida albicans cells and potentially novel antifungal activities. They showed that AgNWs penetrated the biofilm matrix and transported ZnFe2O4 NPs into it, so ZnFe2O4 NPs could damage Candida cells by generating reactive oxygen species (ROS)  who woral., 2016). An addal., 2016) who inval., 2019). Mussinal., 2019). Thakural., 2019). Szerenal., 2019).Paracoccidioides brasiliensis) loaded on PLGA was utilized to treat paracoccidioidomycosis. Male BALB/c mice infected with Paracoccidioides brasiliensis  were used in this study. They also deployed animal models for which a combination of sulfamethoxazole/trimethoprim with the P10 peptide entrapped within PLGA was utilized and observed fulfilling results in some treatments due to P10 elicits, a Th1-like immune response which can control fungal infection . On the other hand, studies on rats disclosed that the oral delivery system of the NPs manifested minimal toxicity and better efficacy compared to fungizone  formulation for ophthalmic drug delivery in an attempt to streamline ocular KTZ delivery. Using this formulation, the researchers observed the enhanced antifungal activity against the standard strain of Candida albicans ATCC 76615 and also reported that these NPs could be used in the treatment of both keratitis and endophthalmitis owing to their ability for transcorneal permeation . Among al., 2010). In 201al., 2014). Other al., 2015). Workinal., 2015). Radwanal., 2015) prepareal., 2017). In anoal., 2017) developeid, 2017).Aspergillus flavus, the researchers found that these NPs can enhance drug residence time, down-regulate inflammation, and release antifungal drugs  is one of the substances used in the production of NPs. Several noticeable advantages of gelatin as NPs are their biodegradable, non-toxic, non-immunogenic biocompatible nature with human tissues as well as their easy crosslinking and potential to modify chemically. Gelatin NPs are delivered to special tissues without compromising drug stability and concentration which is considered a plus point  conditions. Sodium alginate nanospheres of AmB were prepared by the controlled gellification method in this study. Having compared the nanosphere-bound drug with the free drug, the researchers reported that upon using this formulation, a significant reduction in colony-forming units (CFU) in the liver and lungs was observed. This finding, in turn, contributes to a reduction in the total dose required for therapy and, consequently, the degree of drug toxicity -linked β-d-mannuronic acid and α-l-guluronic acid residues . AlginaCandida strains, they reported that these formulations were able to prevent Candida biofilm formation and improve their biocompatibility  (such as iron oxide NPs) are a class of NPs that can be manipulated using magnetic fields and due to magnetic properties, antibiotics included in such complexes can be delivered to specific areas using a magnetic field . Muñoz-ópez 2020).Candida tropicalis, which has a determining role in higher uptake by fungal cells. The researchers used the cornea obtained from the porcine eye to investigate ex vivo corneal permeation which ultimately demonstrated the effective penetration of FLZ-CNGs into the deeper sections of the cornea  which are natural polymeric NPs with great potential in the field of nanotherapeutics. Chitin is an excellent material for the synthesis of nanoparticles as a natural renewable resource, demonstrating biodegradable, biocompatible, and non-toxicity traits .In vitro antifungal activity was performed by researchers against Candida albicans using the M27-A3 (2008) guidelines of clinical and laboratory standards institute indicating that AmB-loaded NPs have an antifungal potential similar to that of the free drug. After investigating ocular pharmacokinetics, they reported the prolonged residence time on the ocular surface. On the other hand, in a corneal penetration study, they observed the penetration of NPs into the cornea  are highly regarded in the drug delivery system these days. To this end, Chhonker et al. made use of AmB loaded L/CS NPs prepared by ionic gelation and achieved the prolonged release of AmB with a reduction in the dosage of pure drug and the consequent side effects. In the in vitro study of Candida albicans and Aspergillus fumigatus, the researchers perceived the potential antifungal efficacy by preparing AmB loaded L/CS NPs and similarly, based on the in vivo study conducted in New Zealand on albino rabbit eyes, improved bioavailability compared to the free drug was noted. Thus, they reported that this formulation holds a higher ocular tolerance than the marketed formulation, however, further studies are necessary for commercial applications . In 201al., 2013). In anoal., 2018). Paul aal., 2018). Sandhyal., 2018) also obal., 2020).in vitro studies, the prolonged release of natamycin was reported and subsequently, the in vivo conditions yielded highly satisfactory outcomes as well  is one of the synthetic biodegradable polymers for drug delivery and biomedical applications. PCL NPs depict low particle size and high flexibility. PCL-based NP formulations have slow release due to high crystalline nature. Also, these NPs have less water permeability and consequently a very slow degradation rate . In theal., 2014) also acal., 2018). All stal., 2018; Ahsan aal., 2018; Amaral al., 2018; Bhalekaal., 2018; Butani al., 2018; Cassanoal., 2018; Chandasal., 2018; Chhonkeal., 2018; Das et al., 2018; El-Housal., 2018; Fu et aal., 2018; Füredi al., 2018; Gupta eal., 2018; Hussainal., 2018; Jain etal., 2018; Kenechual., 2018; Khan etal., 2018; Kumar aal., 2018; Liu et al., 2018; Lucena al., 2018; Mohammeal., 2018; Mohantyal., 2018; Muñoz-Eal., 2018; Mussin al., 2018; Niemiroal., 2018; Pandey al., 2018; Paul etal., 2018; Radwan al., 2018; Sandhyaal., 2018; Sangeetal., 2018; Sanna eal., 2018; Song etal., 2018; Souto eal., 2018; Souza eal., 2018; Szerencal., 2018; Thakur al., 2018; Tutaj eal., 2018; Vásquezal., 2018; Xu et aal., 2018).in vitro toxicity models. It has been generally agreed that NPs interfere with either assay materials or detection systems. Based on multiple applications in multiple areas, human exposure to NPs, both intentionally and unintentionally, is inevitable. Before being considered for human application, all nanoproducts are subjected to toxicological studies and for this purpose, several experimental studies have been carried out. To meet this regulatory requirement, some toxic effects of nanomaterials have been evaluated, but according to reports, the toxicological data derived so far are conflicting and inconsistent . Differal., 2020). Toxicoal., 2012). Howeveal., 2012). Moreoval., 2012).Today, fungal infections gain ever-increasing significance given the growing annual incidences and mortality rate emanating from such infections. Although today the antifungals which are effective in treating these fungal infections are commercially bountiful, researchers have sought ways to optimize the existing drugs thanks to limited penetration through tissue, poor aqueous solubility, reduced bioavailability, drug efficacy and drug resistance, side effects, and poor drug pharmacokinetics. Hence, during the last two decades, the use of NPs drug-delivery systems to enhance the scope of antifungal functions has gained a prominent place in antifungals' optimization. The main merits of these NPs are their small size and large surface area that make them ideal candidates for various applications and able to overcome the limitations of the existing drugs, though partially. Yet, considering all the studies conducted in this field, except the few cases mentioned , none of these formulations are commercially available largely owing to preclinical problems concerning the studies and clinical trials as well as the limitations surrounding the utilization of these NPs.The study was approved by the ethics committee of Tabriz University of Medical Sciences (Grant no. IR.TBZMED.VCR.REC. 1398.103).This work was supported financially by Research Vice-Chancellor, Tabriz University of Medical Sciences, Tabriz, Iran.The authors declare that they have no conflict of interest.


Current neurophysiological constructs postulate that there are two main mechanisms bywhich physical activity may amplify the neural drive to the inspiratory muscles, leading toexertional dyspnea: (a) by increasing the ventilatory demands and (b) by increasing the effortto breathe at a given level of ventilation. In simple terms, dyspnea depends on “how much” ventilation is required and “how well” such ventilation is achieved, respectively. On a purely descriptiveperspective, (a) can be understood as the quantitative domain of exertional dyspnea, whereas(b) reflects its qualitative properties*. A robust body of evidence indicatesthat the former strongly depends on the extra ventilation required to overcome an enlargedphysiological dead space, while the latter is related to neuromechanical dissociation arising at higher operatinglung volumes. Not surprisingly, therefore, the highest dyspnea scores on exertion are reported byCOPD patients depicting (a) poor gas exchange efficiency . Amongst them, obesity assumes prominence due to its high prevalence. Notably, obesity may affect “how much”7 and “how well” ventilation is performed on exertion. For instance, moving a large mass against gravity increases the metabolic cost of work On the other hand, if functional residual capacity (FRC) is downwardly displaced by theincreased weight of the chest wall with little change in TLC, the volume available for VTexpansion in the presence of expiratory flow limitation increases, that is, higher inspiratorycapacity (IC).11 Rather paradoxically,therefore, mild-moderate obesity may yield a mechanical advantage to the obese patient withCOPD.13 In fact, this has beenpreviously demonstrated by Ora et al. as the deflating effects of mild-moderate obesitypostponed the attainment of critically high inspiratory constraints. In more severe obesity, however, TLC may decrease, potentially decreasing IC despite alow FRC. To make things even more complex, fat distribution has a major impact on theseinter-relationships since the android/central pattern of obesity has a larger lowering effecton lung volumes (including TLC) compared to the gynecoid/peripheral pattern. Breathing at excessively low lung volumes, in turn, may predispose to the closure ofsmall airways, leading to ventilation/perfusion mismatch and gas trapping.There are several factors, however, that may modulate the severity of exertional dyspnea at agiven level of resting functional impairment in patients with COPD.Chronic Respiratory Disease, Zewari and colleagues dealt with another feature of the obesitydyspnea conundrum in COPD: the influence ofexercise modality. Despite the lack of direct comparisons between walking versus cycling andthe dearth of physiological measurements on exertion, the authors conclude that evenmild-moderate obesity may have a detrimental effect on exercise tolerance and dyspnea in thesepatients. Some caution, however, should be exerted to interpret their findings; for instance,only the 6-min walk work  was higher in the obese group since therewere no between-group differences in the 6-min walking distance. Although statisticallysignificant, 010 Borg dyspnea scores on exercise cessation were only slightly higher in obesesubjects (average 4 compared to 3.2). Interestingly, anthropometric markers of abdominaladiposity in the obese group appeared to significantly worsen dyspnea and exercise tolerance,likely due to its greater mechanical consequences.In the current issue of  Walking is characteristically associated with greater ventilatory requirements comparedto cycling due to (a) higher  (b) additional ventilatory stimuli secondary to upper limbs’ movement, and, in some patients, (c) greater exercise-induced hypoxemia resulting from lowermixed venous O2 pressures and a delayed onset of hyperventilation to compensate formetabolic acidosis. The abdominal expiratory muscles are involved in postural actions during walking; beingless available to decrease FRC and increase IC compared to cycling. If the patients anchor their accessory inspiratory muscles by firmly holding thehandlebars, larger IC can be reached on cycling than walking. Zewari and colleagues argue that the negative consequences of mild-moderate obesity on dyspnea during walkinghave more than eclipsed its putative salutary effects previously described by Ora et al. inresponse to cycling. As mentioned, however, these assertions were based on indirect comparisons betweendifferent populations. Dynamic hyperinflation was only inferred by changes in IC induced byshort (20 s) increases in resting breathing frequency at rest, that is, it was not measuredduring exercise. Moreover, other mechanisms of exercise intolerance which are more prevalentin the obese, such as severe deconditioning and cardiovascular co-morbidities, may have variedsubstantially in each individual study.Why would walking cause greater dyspnea than cycling in the obese patient? Is there an equilibrium point where the putative beneficial effects of mild-moderateobesity on lung mechanics compensate for the negative consequences of increased ventilatorydemands? If so, this is likely to vary with exercise intensity and whether there are othersources of ventilation stimuli, such as a low PaO2 or early metabolic acidosis, ornot. Are the negative effects of android/central obesity on exertional dyspnea greater onwalking than cycling? Are they even greater in subjects with small trunks, that is, shorterpatients? Importantly, most studies looked at patients with mild-moderate obesity with only afew patients in the morbid obesity range.There remains, therefore, several unanswered questions on this topic. For instance, it isconceivable that the negative consequences of increased ventilatory demands associated withwalking are particularly pronounced in patients with higher “wasted” ventilation in thephysiological dead space. future studies should consider not only the severity of obesity as estimated by bodymass index (BMI) but, crucially, body fat distribution in a sizable number of men and womenshowing a large range of resting functional abnormalities. The same subjects should undergowalking and cycling exercise: their sensory and physiological responses (including operatinglung volumes) compared at iso-work rate and iso-ventilation. Ideally, measurements should berepeated after weight loss to determine the directional changes on the quantitative andqualitative domains of exertional dyspnea. Advancing the knowledge on the seeds and consequences of activity-related dyspnea inthe obese patient with COPD are likely to directly impact the care of this ever-growingpatient subpopulation.To answer these and other questions,


Patel et al., the prevalence of atrial tachyarrhythmias and atrial fibrillation (AF) in particular, has been increasing in the general US population with an estimated 59.7 million cases of AF or atrial flutter reported in 2019  annually. SCD instigated by cardiac arrhythmias accounts for over 300,000 deaths annually in the US alone , renal denervation or spinal cord stimulation has been shown to reduce infarct size, modulate cardiac alternans, demonstrate anti-arrhythmic and anti-inflammatory effects  based techniques for predicting cardiac abnormalities, and also offer a perspective on bridging the gap between basic and clinical science for the successful translation of promising anti-arrhythmic pacing strategies. Their review provides a detailed overview of traditional clinical methods used for arrhythmia diagnosis, such as Holter monitoring, cardiac mapping, tilt table tests, pharmacological interventions, and implantable devices like pacemakers and cardioverter defibrillators. Additionally, they provide an overview of promising preclinical and clinical studies investigating novel non-linear dynamical pacing modalities for arrhythmia management.In their review article, Ma et al. present results from an interesting study investigating the effect of acute exposure to a hypoxic environment on the cardiopulmonary system. The autonomic nervous system plays a vital role in the acclimatization of the heart to external or internal stimuli. Their study investigated the longitudinal changes and recovery in heart rate variability (HRV) in young healthy adults on exposure to a simulated plateau environment. The participants were subjected to normoxic (~101 kPa) and hypoxic  environments, and the effect of pressure change on HRV was investigated. They demonstrated that participants in hypobaric hypoxia had significantly lower HRV time-domain metrics, substantially higher LF/HF ratio, significantly lower Poincaré plot parameters and significantly reduced refined composite multi-scale entropy curves. Their study shows that exposure to hypobaric hypoxic environment can result in increased heart rates and sympathetic activation.Kulkarni et al. demonstrated that non-invasive, low level tragus stimulation (LLTS), modulates microvolt T-wave alternans in patients with ischemic cardiomyopathy and heart failure. Cardiac alternans has been shown to be a marker of impending arrhythmias and heightened levels of T-wave alternans have been associated with an increased predisposition to ventricular tachyarrhythmias. Prior preclinical studies have demonstrated that inhibition of T-wave alternans could prevent the onset of fatal tachyarrhythmias. In this present study, acute LLTS was performed on 26 patients with ischemic cardiomyopathy (left ventricular ejection fraction <35%) and heart failure. LLTS was performed at 5 and 20 Hz for 15 min, in active patients, both during sinus rhythm and with concomitant atrial pacing at 100 bpm. Their results demonstrate that right atrial pacing at 100 bpm leads to significantly heightened T-wave alternans burden compared to sinus rhythm, with or without LLTS. In addition, acute LLTS at both 5 and 20 Hz was shown to increase T-wave alternans burden. This study highlights the potential of acute LLTS in modulating alternans burden and forms the basis for evaluating the chronic effect of LLTS on T-wave alternans and ventricular tachyarrhythmia burden in this patient population.In another novel application of vagus nerve stimulation, Kuwabara et al. investigated the utility of dorsal root ganglion stimulation (DRGS) in reducing cardiac sympathoexcitation and in turn ventricular arrhythmogenicity. They tested the induction of cardiac sympathoexcitation in swine using ventricular extrastimulation, before and after DRGS. Their data demonstrate that DRGS at both 20 Hz and 1 kHz, decreases pacing-induced activation recovery intervals shortening, reduces arrhythmogenicity by flattening the slope of the electrical restitution curve, and also decreases the Tpeak-Tend/QTc ratio. This study highlights the anti-arrhythmic effects of thoracic DRGS at both low-frequency and high-frequency, demonstrating its potential for countering cardiac excitability during sympathetic overdrive.Sympathetic overdrive has been shown to be associated with ventricular tachyarrhythmias and SCD. In an exciting new study, In summary, the collection of articles in this dedicated Research Topic, provides a comprehensive assessment of the contemporary treatment modalities for cardiac arrhythmia prediction, prevention and control. Moreover, these papers present novel promising neuromodulation approaches for treatment of cardiovascular diseases that have the potential to push traditional boundaries and radically improve cardiac patient care. Technological advancements in signal processing, the advent of AI-based diagnostic approaches and the exponential rise in big data engineering have ushered in a new era of medical science. The novel dynamical approaches for rhythm management being investigated, point to an extremely promising future for cardiac pacing therapies. In conjunction, real-time remote patient monitoring and telehealth have enabled access to healthcare for patients in underserved and isolated rural areas while simultaneously expanding accessibility for urban patients as well (Kulkarni et al., All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


HTT) gene, which results in the production of a pathogenic mutant HTT protein (mHTT). Although there is no cure at present for HD, a number of RNA-targeting therapies have recently entered clinical trials which aim to lower mHTT production through the use of antisense oligonucleotides (ASOs) and RNAi. However, many of these treatment strategies are non-selective in that they cannot differentiate between non-pathogenic wild type HTT (wtHTT) and the mHTT variant. As HD patients are already born with decreased levels of wtHTT, these genetic therapies may result in critically low levels of wtHTT. The consequence of wtHTT reduction in the adult brain is currently under debate, and here we argue that wtHTT loss is not well-tolerated at the synaptic level. Synaptic dysfunction is an extremely sensitive measure of subsequent cell death, and is known to precede neurodegeneration in numerous brain diseases including HD. The present review focuses on the prominent role of wtHTT at the synapse and considers the consequences of wtHTT loss on both pre- and postsynaptic function. We discuss how wtHTT is implicated in virtually all major facets of synaptic neurotransmission including anterograde and retrograde transport of proteins to/from terminal buttons and dendrites, neurotransmitter release, endocytic vesicle recycling, and postsynaptic receptor localization and recycling. We conclude that wtHTT presence is essential for proper synaptic function.Huntington disease (HD) is a monogenic disease that results in a combination of motor, psychiatric and cognitive symptoms. HD is caused by a CAG trinucleotide repeat expansion in the huntingtin ( Huntington disease (HD) is an autosomal dominant neurodegenerative disease that results in a triad of motor, psychiatric and cognitive symptoms, and has an estimated prevalence of 13.7 per 100,000 in the general population . AlthougHTT gene , which encodes the large, 348 kDa protein huntingtin (HTT)  . CAG repin (HTT) . Longer in (HTT) . Wild-tyin (HTT) . The N-tin (HTT) . An essein (HTT) . Heterozin (HTT) . Additiolifespan . The essWild type HTT has been shown to act as a scaffolding protein, functioning to stabilize intracellular cargo onto molecular motors to streamline fast axonal transport and to assist in anchoring receptors at the plasma membrane . As we wHTT mRNA aim to reduce the production of the mutant protein variant; however, many of these options will also decrease the production of wtHTT. Furthermore, there is evidence that pathogenic exon 1 fragments can result from incomplete splicing at the pre-mRNA level, thereby evading the mRNA-targeting approaches , RNAi and small molecules, while those targeting DNA include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR-Cas9. Each of the various strategies have specific benefits as well as drawbacks which have been reviewed recently . As an eproaches .RX or RG6042) was recently halted early in phase III. Unfortunately, tominersen was no more effective than placebo when administered every 16 weeks, and actually worsened motor and cognitive symptoms when administered every eight weeks  also make these therapeutics particularly desirable. ASOs can penetrate cell membranes without the need for an accompanying viral vector and can be delivered to the brain through intrathecal injections into the spinal cord. The Roche GENERATION HD1 clinical trial using the non-selective ASO tominersen (previously known as HTTht weeks . The recht weeks .HTT (IT15) gene. Precise silencing of the mutant allele using gene-editing techniques such as CRISPR-Cas9 would halt mHTT transcription at its source; however, gene editing therapies require invasive delivery systems and are largely irreversible. Human clinical trials for DNA-targeting HD therapies are yet to be announced, although initial rodent studies have shown promising results  firing, exhibits poor temporal coordination with AP firing, or occurs independently of AP firing, respectively. The different modes of presynaptic release are associated with overlapping but distinct underlying mechanisms, and research is ongoing to fully understand how presynaptic release is regulated and maintained at different synapses throughout the healthy brain . The comA finite number of SVs exist at presynaptic release sites. During neural activity, SVs release their contents by fusing with the plasma membrane, and the pool of SVs available within a presynaptic neuron can be rapidly depleted during sustained neural activity. For example, at the CA3-CA1 synapse in the hippocampus, it is estimated that approximately 30 seconds of neural activity at a physiologically relevant firing rate of four hertz is sufficient to completely deplete the presynaptic supply of glutamate SVs . To mainFlox/+), restored the ADAM10/piccolo interaction and replenished SV stores  also position wtHTT to influence presynaptic transmission. When the wtHTT/HIP1 interaction was first described, it was recognized that disrupting this interaction could negatively impact the integrity of the cytoskeleton . A few yTogether, the aforementioned studies suggest that wtHTT loss of function (LOF) may disrupt presynaptic homeostasis by impairing clathrin-mediated SV recycling. A direct role of wtHTT in SV endocytosis was demonstrated recently when self-deliverable, cholesterol-conjugated siRNAs were used to knock down wtHTT expression in cultured neurons . In thisIn sum, wtHTT interacts with key presynaptic proteins that regulate SV endocytosis, and wtHTT LOF slows the rate of SV recycling following a period of evoked neural activity. It will be of interest for future studies to further pinpoint the mechanisms underlying wtHTT’s role in SV recycling and to determine how much wtHTT reduction in the adult brain can be safely tolerated before SV replenishment rates are negatively impacted.Drosophila  and retrograde (toward the cell body) transport of various molecular cargoes, respectively . Both paransport . In addiransport , 2003. Transport , and BDNransport . Interesransport .Numerous post-translational modifications (PTMs) of wtHTT have been shown to be essential to its role in vesicular transport. For example, serine 421 (S421) on wtHTT has been identified as an important phosphorylation site that mediates axonal trafficking; phosphorylation of wtHTT at S421 recruits kinesin-1 and promotes anterograde transport of BDNF-containing vesicles whereas S421 dephosphorylation favors retrograde transport following kinesin-1 detachment . On the Impaired axonal trafficking can have profound functional consequences that extend well beyond the aforementioned reduction in BDNF delivery to the striatum. Efficient trafficking of a variety of cargo both to and from synaptic compartments is essential for the maintenance of synaptic homeostasis. For example, wtHTT loss interferes with the delivery of large dense core vesicles (DCV), which carry neurotrophins and neuropeptides, to release sites . GrowingDrosophila, knocking out the Drosophila homolog of wtHTT was found to slow axonal trafficking of synaptotagmin-containing SVs  is required to bring the proper release machinery to the presynapse. Newly synthesized SVP delivery works together with endocytosis, albeit at a slower rate, to contribute to the maintenance of presynaptic SV supply . SVPs arning SVs . More retobrevin . Rab4 istobrevin . The bidvesicles . TogetheIn recent years, it has become clear that autophagy is more than a simple housekeeping process that rids the cell of unwanted materials. In fact, autophagy is being increasingly recognized as a major contributor to synaptic function, which has been recently reviewed elsewhere and will not be extensively covered in the present review . SynapseIn addition to the myriad ways in which wtHTT positively influences presynaptic neurotransmission as discussed above, ample evidence exists to suggest that wtHTT is equally as important to proper postsynaptic function. In this section, we discuss how wtHTT is involved in the bidirectional transport of essential synaptic cargoes between the soma and dendritic tree, postsynaptic receptor clustering and subcellular localization, as well as spine stabilization and synaptic plasticity. Some of ways in which wtHTT can influence postsynaptic function are depicted schematically in A receptors to postsynaptic sites at inhibitory synapses (A receptors and decreased the amplitude of miniature inhibitory postsynaptic currents (mIPSCs). Interestingly, wtHTT/HAP1/KIF5 protein complexes also deliver AMPA receptors to postsynaptic sites at excitatory synapses. In cultured neurons, wtHTT increased the strength of the interaction between HAP1 and KIF5 motors, promoting the transport GluA2-containing vesicles along microtubules to reach postsynaptic sites. Interfering with the protein complex, either through mHTT presence or by knocking down HAP1 or KIF5, reduced AMPA receptor trafficking and decreased mEPSC amplitude. Overexpressing wtHTT had the opposite effect, increasing mEPSC amplitude at excitatory synapses , assists the delivery of GABAsynapses . Disruptsynapses . In addisynapses . For exasynapses , and it synapses .Glued subunit of dynactin and the adaptor protein AP-2. The AP-2-mediated retrograde transport of BDNF/TrkB autophagosomes was shown to bring active TrkB to the soma where it promoted neuronal complexity and protected against neurodegeneration , wtHTT acts as a positive modulator of palmitoylation, a PTM known to affect the subcellular localization and clustering of numerous synaptic proteins. For example, palmitoylation of PSD-95 targets this important postsynaptic scaffold to synaptic sites, and thereby regulates glutamate receptor clustering and activity-dependent synaptic plasticity . Direct Through interaction with PSD-95, wtHTT forms a complex with NMDA and kainate receptors, and decreasing the interaction between HTT and PSD-95 can sensitize NMDARs . Thus, iIn addition to palmitoylation discussed above, both wtHTT and mHTT are subject to numerous other PTMs including proteolysis, phosphorylation and myristoylation, to name a few . ConsideIn addition to the previously discussed role of ADAM10 in presynaptic SV regulation, this metalloproteinase is also found in dendrites and spines and is known to play a major role in postsynaptic function. wtHTT inhibits ADAM10, and hyperactive ADAM10 can result from mHTT presence or from wtHTT loss . HyperacSynaptic plasticity deficits have been observed in numerous mouse models of HD  as well It is indisputable that wtHTT is essential for development, as its knockout is embryonic lethal . FurtherConditional wtHTT models also clearly demonstrate that wtHTT is necessary for proper synapse development, function, and stability. Interestingly, early hyperexcitability that cannot be maintained has been reported in HD . In the in vivo studies have been performed that reduce wtHTT to varying amounts in the brain, a clear consensus has yet to be reached  does not negatively impact motor function nor does it cause any obvious signs of neurodegeneration, gliosis or inflammation . In the HTT allele that reduces its expression is associated with an earlier disease onset (HTT was floxed while the other was knocked out. In other words, these mice developed with half the normal levels of wtHTT before wtHTT was depleted by tamoxifen injection in adulthood at 3, 6, and 9 months of age. When wtHTT was depleted in adulthood in these mice, the authors observed a significantly reduced lifespan, impaired motor performance on the rotarod, various behavioral abnormalities (as assessed by the SHIRPA scale described previously Huntington disease mutation carriers almost always carry one copy of each the mutant allele and the wild-type allele. As a result, wtHTT levels are reduced throughout development and will be reduced further with any of the non-selective HTT reduction strategies. wtHTT reduction during development is likely to exert a negative effect on its own, as wtHTT heterozygous mice exhibit motor and cognitive deficits as well as neurodegeneration . Interesse onset . Recentlin vivo, many putative emerging synaptic deficits are likely to go unnoticed in behavioral assessments that occur within a few months of wtHTT knockdown. After all, an excessive number of CAG repeats — only seen in the most severe cases of juvenile HD — is required to produce a HD-like phenotype within the lifespan of a rodent. In HD, synaptic deficits emerge before cells die and before a behavioral phenotype is evident (Many of the studies discussed in this review demonstrate a clear role for wtHTT in synaptic function; for example, by complexing with essential synaptic proteins or by facilitating the delivery of synaptic cargoes from the cell body to pre- and postsynaptic compartments. In studies that reduce wtHTT  evident . Here, w evident . Iron ho evident , even th evident . Similar evident . Thus, w evident . By obtaJB, EH, and MP wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


E. coli and possibly across numerous other bacteria, based on the stoichiometry of interacting protein pairs (from proteomic studies) and their conservation across bacteria. We present a list of 339 protein-protein interactions involving glycolytic enzymes but predict that ~70% of glycolytic interactors are not present in adequate amounts to have a significant impact on glycolysis. Finally, we identify a conserved but uncharacterized subset of interactions that are likely to affect glycolysis and deserve further study.Glycolysis is regulated by numerous mechanisms including allosteric regulation, post-translational modification or protein-protein interactions (PPI). While glycolytic enzymes have been found to interact with hundreds of proteins, the impact of only some of these PPIs on glycolysis is well understood. Here we investigate which of these interactions may affect glycolysis in  Proteomics has produced invaluable data about the composition, abundance, and function of all proteins in cells and organisms . SimilarGlycolysis is the primary central carbon metabolic pathway responsible for turning glucose into pyruvate , besides producing ATP and other metabolites . GlycolyE. coli and other bacteria, there are few comprehensive analyses of the interface of the protein interactome and the metabolome in this or other species  studies that provided the PPI data  and ptsH  were found in the abstract of Rodionova et al. /carbohydrate metabolic process [GO:0005975]- being the most common with 27 interactors. Remaining 210 interactors showed a wide range of GO metabolic terms including ribosomal large subunit assembly (translation), ATP biosynthetic process, L-proline biosynthetic process (amino acid metabolism) etc. There were 25 proteins which did not show any GO metabolic terms C,D.For instance, Eno had 35 metabolic protein interactors, 43 non-metabolic protein interactors, 7 interactors which did not show any GO terms and 5 uncharacterized proteins. The most-common metabolic GO terms among enolase’s interactors were cellular response to DNA damage stimulus [GO:0006974] and mRNA catabolic process [GO:0006402]. Interestingly, enolase had more non-metabolic interactors, and the most over-represented (high-level) GO terms dealt with regulation of cell shape [GO:0008360], peptidoglycan biosynthetic process [GO:0009252] and cell wall organization [GO:0071555] (43 interactors).The evidence-based analysis reflected the pattern we saw in the enzyme vs. non-enzyme distribution, with most enzymes having metabolic interactors. FbaB was the only glycolytic enzyme that had only non-metabolic interactors, namely DnaJ (chaperone/heat shock protein J).Overall, more metabolic than non-metabolic interactors were found in the glycolytic interactome, as expected, but the substantial number of non-metabolic interactors indicates an extensive crosstalk between glycolysis and other processes in bacterial cells.We used the KEGG database to determine the pathway characteristics of the interactors E,F. Our While glycolysis is an important central pathway, it is not absolutely essential, as long as cells can obtain energy from sources other than glucose, such as amino acids. However, if glucose is the only carbon source, glycolysis becomes an absolutely essential pathway, hence it is conditionally essential. We wondered if glycolytic enzymes interact with other essential proteins or whether these interactors are dispensable.We evaluated if a protein is encoded by an essential gene using the Database of Essential Genes (DEG). Then, we counted the number of essential genes and non-essential genes among the interactors of glycolytic enzymes. Uncharacterized proteins are not essential, as expected. These proteins did not have an EC number (except 1 protein: YegV), and did not show metabolic terms from GO or KEGG (metabolic pathways).only non-essential proteins as interactors  levels. Although there is little experimental data supporting this assumption for in vivo levels of enzymes, we investigated the expression levels of glycolytic interactors under different conditions as described by Schmidt et al. .We normalized the abundance values of all interactors to the level of the glycolytic enzyme (set to 1). In general, the abundance of most interactors is lower than that of their corresponding glycolytic enzymes . That isMetabolic pathways are related to many diseases, and many therapeutic applications cannot be properly developed if an adequate understanding of their regulation is not available. However, research has focused mainly on allosteric regulation, gene regulation and post-translational modifications. Protein-protein interactions (PPIs) can also be a very important route in terms of affecting metabolic enzymes ,9,10. HeIn order to get a more comprehensive picture of glycolytic PPIs, we visualized the glycolysis PPI network . The netThe flux of metabolic reactions is determined by the concentrations of enzymes and substrates, but also by their activities and their rate-limiting steps. Hence, we explored the abundance of the glycolytic proteome with respect to its interactome . We founDespite the almost universal conservation of glycolysis throughout all domains of life, many of the interactors considered in our analyses are much less well conserved . While cE. coli and other bacteria  activities of these proteins. For example, knocking out the binding site on rne (Ribonuclease E) may increase or decrease the metabolic rate of