Melanoma is a complex and aggressive cancer type that contains different cell subpopulations displaying distinct phenotypes within the same tumor. Metabolic reprogramming, a hallmark of cell transformation, is essential for melanoma cells to adopt different phenotypic states necessary for adaptation to changes arising from a dynamic milieu and oncogenic mutations. Increasing evidence demonstrates how melanoma cells can exhibit distinct metabolic profiles depending on their specific phenotype, allowing adaptation to hostile microenvironmental conditions, such as hypoxia or nutrient depletion. For instance, increased glucose consumption and lipid anabolism are associated with proliferation, while a dependency on exogenous fatty acids and an oxidative state are linked to invasion and metastatic dissemination. How these different metabolic dependencies are integrated with specific cell phenotypes is poorly understood and little is known about metabolic changes underpinning melanoma metastasis. Recent evidence suggests that metabolic rewiring engaging transitions to invasion and metastatic progression may be dependent on several factors, such as specific oncogenic programs or lineage-restricted mechanisms controlling cell metabolism, intra-tumor microenvironmental cues and anatomical location of metastasis. In this review we highlight how the main molecular events supporting melanoma metabolic rewiring and phenotype-switching are parallel and interconnected events that dictate tumor progression and metastatic dissemination through interplay with the tumor microenvironment. Genetic and non-genetic intra-tumor heterogeneity define the coexistence of phenotypically and metabolically different cancer cell subpopulations within the same tumor and are one of the major features of tumor complexity . It is iMetastasis, the major cause of cancer-related death, is a very inefficient process. For a metastatic lesion to occur, cancer cells undergo a series of phenotypic changes that allow the acquisition of migratory capacity, the ability to survive in circulation and at the destination tissue, and the potential to proliferate when the seeding organ has been reached. Although millions of cancer cells are able to escape from the primary tumor into the blood or lymph, very few succeed at initiating a secondary lesion in a distant organ .To escape from and survive outside of the primary tumor, invasive tumor cells require metabolic flexibility, first to fuel the high energy demanding migratory process , and secMelanoma, the deadliest form of skin cancer, arises from the oncogenic transformation of cells belonging to the melanocytic lineage  and reprConsidering that cell metabolism is tightly associated with both phenotypic identity and the adaptation to local and systemic microenvironmental factors, it is not surprising that changes in the tumor microenvironment (TME) have an impact on tumor metabolism and melanoma cell fate. Furthermore, recent evidence suggests that other factors such as anatomical location, which might itself be subjected to specific microenvironmental cues, have an impact on melanoma cell features .In this review we focus on the metabolic traits that characterize the different phenotypic states of melanoma cells and the metabolic reprogramming that might dictate the transitions between states that drive melanoma progression and metastasis. Since the role of glucose and glutamine metabolism in melanoma has been widely discussed in recent well-documented reviews , here weMelanoma is characterized by high levels of genetic heterogeneity and displays one of the highest somatic mutational burdens among all cancers . Some ofAlthough the study of these genetic alterations is essential for the understanding of the mechanisms involved in melanoma initiation, evidence suggests that the melanoma mutational landscape is not sufficient to explain why phenotypic heterogeneity and metastatic dissemination occur. Although oncogenic mutations might facilitate the metastatic process , they arGenetic intra-tumor heterogeneity may impose tumor metabolic heterogeneity. Indeed, most human tumors present somatic mutations, many of which are likely to affect metabolism . A compein vivo melanoma cells may switch between these states, and since tumors show evidence for both proliferative and invasive cell types it is likely that microenvironmental conditions drive this transcriptional switching and determine cell plasticity  and the mono-unsaturated fatty acids (MUFAs) is also dependent on MITF. The SFA:MUFA ratio, essential for proper membrane biogenesis during cell proliferation, is directly regulated by the activity of the oxygen and iron-dependent lipogenic enzyme stearoyl Co-A desaturase 1 (SCD1), which converts long chain SFAs to MUFAs . In melaIn addition to promoting FA synthesis, a further functional role of MITF in regulating energy metabolism to sustain melanoma proliferation has been observed after treatment with diclofenac and lumiracoxib, two commonly used therapeutic agents that decrease melanoma proliferation by impairing oxidative and glycolytic pathways through MITF downregulation . RelatedMoreover, MITF directly activates the transcription of the peroxisome proliferator-activated receptor gamma 1-α (PPARGC1A or PGC1α) , a transAnother direct transcriptional target of MITF is the gene RRAGD, which encodes RagD, a protein implicated in the activation of the mTORC1 complex, a key amino acid-regulated metabolic hub that promotes protein synthesis and suppresses autophagy. By regulating RagD protein expression, MITF controls the recruitment of mTORC1 to lysosomes where it senses amino acids and consequently controls its activity. However, although regulation of RagD has been reported in Hela cells engineered to ectopically overexpress MITF , it has MITF, like TFEB, is a regulator of lysosomal biogenesis mediated by activation of AMPK, a kinase activated by high AMP:ATP levels. MITF can bind to the promoters of lysosomal and autophagosomal genes and plays a crucial role in the regulation of autophagy in melanoma cells in response to mitochondrial stress and starvation . ConsistCollectively, the current evidence strongly suggests that MITF works in melanoma as a central metabolic sensor that couples and integrates cell-extrinsic nutritional signals with cell-intrinsic metabolic responses to drive melanoma cells towards specific phenotypes that better guarantee melanoma survival under dynamic microenvironmental conditions.MITF locus , essential for FA and cholesterol biosynthesis . Indeed,de novo generation of FAs from other available metabolites, increasing evidence shows that transformed cells have also an increased capacity to uptake FAs from external sources  programs. A significant enrichment in genes involved in FA uptake, such as CAV1 or the FA transporter CD36, was found in metastatic tumors, revealing also a significant negative effect of FA uptake on patient survival rates . These din vivo, the invasive state induced by treatment with BRAF inhibitors observed by Along the same lines, metastasis-initiating cells in melanoma and other cancer types show as a common feature the overexpression of CD36 . Consequde-novo FA synthesis might determine the efficacy of targeting CD36 to stop cancer progression , can increase the metastatic capacity of melanoma by decreasing oxidative stress and ferroptosis in cells migrating through the lymphatic system . MechaniThe current evidence therefore suggests that while FA internalization may be a metastatic trigger, it is not exclusively linked to CD36. In this regard, the participation of a different FA transporter, FATP1/SLC27A1, has also been shown to be crucial for melanoma progression and dissemination. Melanomas significantly overexpress this transporter on the tumor cell surface to uptake adipocyte-derived lipids, as its pharmacological inhibition impairs lipid internalization, thereby reducing tumor growth and invasion . FurtherRegarding the usage of FA by melanoma cells, consequent to their increased uptake, it seems clear that the overexpression of FA transporters leads to a subsequent increase of mitochondrial FA Oxidation (FAO), and the coupling of both events promotes melanoma aggressiveness , suggestin vitro in metastatic melanoma cells that instead tend to use FAs to generate more complex structural and signaling lipids, such as ceramides  and the subsequent phosphorylation of one of its transducers, the eukaryotic translation Initiation Factor 2 alpha (p-eIF2α). This in turn induces a shut-down of global translation, but also promotes melanoma invasion and metastasis . ImportaIn a cancer cell, if on the one hand the ISR attenuates global mRNA translation to activate an alternative proteome to cope with oncogenic and microenvironmental stress, on the other hand activation of the mTOR pathway increases translation to match the proliferative demand. It is therefore plausible that the mammalian (or mechanistic) Target of Rapamycin mTOR pathway, a key sensor and effector of nutrient signaling as well as protein and lipid biosynthesis, plays an important role in melanoma progression, by itself or through a crosstalk with the ISR.While we focus here on the role of mTOR regulation in melanoma, for a more comprehensive overview of the signaling pathways related to mTOR in physiological and pathological conditions we refer the reader to the recent reviews from mTOR is the catalytic subunit of two distinct multi-protein complexes, mTORC1 and mTORC2. mTORC1 is largely dedicated to amino acid sensing, it is inhibited by AMPK, the energy sensor of the cell, and it is activated by PI3K and MAPK signaling downstream from tyrosine kinase receptors (RTKs). mTORC2 also responds to PI3K and growth factor signaling and is activated by low glucose.Mechanistically, in response to growth factors, nutrients, or oncogenic activation, mTORC1 activates protein synthesis on one side by phosphorylating the kinase (S6K) of ribosomal protein S6 , and on mTORC2’s primary role is through the regulation of AKT  and membIn melanoma, the PI3K pathway is activated through several mechanisms, both genetic and non-genetic, leading to mTORC1 activation . In BRAFTo complicate the picture, despite the apparent opposing effects of the ISR and mTORC1 signaling pathways, an intimate interconnection is emerging since 4E-BP1 is a transcriptional target of the ISR effector ATF4 . In the Pathria and colleagues demonstrated that, in melanoma, adaptation to Lactate Dehydrogenase A LDHA inhibition, meant to disrupt the “Warburg effect,” is mainly associated with activation of the ISR, which in turn increases glutamine uptake and mTORC1 activation .in vitro and in human melanoma samples  or as recurrence after targeted therapy  . While tterparts . Togetheterparts . Indeed,ergetics . Furtherergetics .Melanoma cells bear the highest expression of PGC1α, compared to other cancer cell lines , and higOn the other hand, melanomas expressing high PGC1α basal levels show increased metastatic capacity when PGC1α is suppressed . TherefoIt is possible that the double role of PGC1α in promoting tumor growth and suppressing metastasis is dependent on the phenotypic state of the melanoma cell, and therefore its metabolic requirements. In the proliferative MITF-high state PGC1α favors oxidative metabolism, a proficient and economic way of supplying energy. In the invasive MITF-low state PGC1α depletion rewires melanoma cells towards metabolic dependency on HIF1α-mediated glycolysis and glutamine usage . FinallyRemarkably, PGC1α also exerts a ROS scavenging effect through the induction of many ROS-detoxifying enzymes, such as SOD2 and GPX1 . Indeed,On the other hand, it appears that oxidative stress could promote cell migration and invasion in lung cell carcinoma  as well Since ROS are drivers of metabolic stress, a role of the ISR in regulating ROS detoxification has been elucidated, whereby ATF4 is capable of activating an antioxidant response through transcription of the major antioxidant enzymes HO-1  and glutFinally, a role of mitochondria dynamics is emerging in regulating melanoma tumorigenesis and metastasis. As an example, markers of mitochondria fission and fusion such as dynamin-related protein 1 (DRP1), mitochondrial fission protein 1 (FIS1), and mitofusins are upregulated in patient tumor samples compared to their healthy counterparts and are significantly associated with metastasis . MoreoveA major driver of therapy resistance is phenotypic heterogeneity arising as a consequence of adaptation to microenvironmental cues, including nutrient limitation, inflammation, and crucial in the clinical setting, exposure to chemotherapy itself . Facing Further research is therefore necessary to determine whether using therapies targeting lipid metabolism in combination with immunotherapy may be effective by promoting the desirable responses while avoiding the invasive and metastatic effects that are also associated with, for example, long-term SCD inhibition or stimulation of FAO.Despite being a highly inefficient process, metastasis is the main cause of cancer death for a wide variety of cancer types. Melanoma represents a paradigm for understanding disease progression including metastatic dissemination, since despite its low frequency there are excellent markers that highlight specific phenotypic subpopulations. In this review we have described many of the recently identified mechanisms of metastatic dissemination which rely on metabolic rewiring and the main pathways regulated by and regulating it . We shedHowever, specifically targeting the metabolic vulnerabilities of cancer cells without affecting the healthy population is a major challenge that needs to be addressed. Furthermore, phenotypic heterogeneity poses a challenge also for targeting metabolic pathways. Indeed, the coexistence of metabolically different populations within the same tumour may hamper the complete eradication of the tumour mass when using targeted therapies. The balance between the targetable metabolic pathways and their specificity could provide a new avenue of treatment. Another potentially valuable approach could derive from pharmacologically directed phenotype switching to drive melanoma cells toward a unique specific and targetable metabolic phenotype . While t


In the cell, RNA exists and functions in a complex with RNA binding proteins (RBPs) that regulate each step of the RNA life cycle from transcription to degradation. Central to this regulation is the role of several molecular chaperones that ensure the correct interactions between RNA and proteins, while aiding the biogenesis of large RNA-protein complexes (ribonucleoproteins or RNPs). Accurate formation of RNPs is fundamentally important to cellular development and function, and its impairment often leads to disease. The survival motor neuron (SMN) protein exemplifies this biological paradigm. SMN is part of a multi-protein complex essential for the biogenesis of various RNPs that function in RNA metabolism. Mutations leading to SMN deficiency cause the neurodegenerative disease spinal muscular atrophy (SMA). A fundamental question in SMA biology is how selective motor system dysfunction results from reduced levels of the ubiquitously expressed SMN protein. Recent clarification of the central role of the SMN complex in RNA metabolism and a thorough characterization of animal models of SMA have significantly advanced our knowledge of the molecular basis of the disease. Here we review the expanding role of SMN in the regulation of gene expression through its multiple functions in RNP biogenesis. We discuss developments in our understanding of SMN activity as a molecular chaperone of RNPs and how disruption of SMN-dependent RNA pathways can contribute to the SMA phenotype. Survival Motor Neuron (SMN) is a ubiquitously expressed protein composed of 294 amino acids with multiple domains that provides a platform for RNA and protein binding during its function as molecular chaperone of ribonucleoprotein (RNP) complexes ,2,3. SMNsurvival motor neuron 1 (SMN1) gene located on chromosome 5q13 [SMN2) that differs from SMN1 by a small pool of nucleotides, with the most significant difference being a C-T transition in exon 7. This modification creates a splicing alteration that leads to skipping of exon 7 (SMN∆7) in approximately 90% of SMN2 transcripts, which are therefore unable to generate a full-length SMN protein [SMN is encoded by the ome 5q13 . Humans Mutations or deletions in the SMN1 gene result in spinal muscular atrophy (SMA), a neuromuscular disorder with onset mainly during the early childhood. With a prevalence ranging from 8 to 10 per 100,000 individuals and an incidence of 1:10,000 live births, SMA represents the leading cause of infant death due to a monogenic disease ,10,11. SThe clinical phenotype directly correlates with the number of SMN2 copies present in the genome. Patients with more than three copies of SMN2 usually display milder SMA symptoms, confirming that SMN2 functions as a compensatory mechanism and further associates the loss of fully functional SMN with the SMA phenotype ,14. DecaIn this review, we focus our attention on the pleomorphic role of the SMN complex in RNA metabolism and processing, first examining its various functions and then detailing how their dysfunctions result in the SMA phenotype. We provide a comprehensive overview of canonical and recently discovered functions of the SMN complex, covering basic molecular concepts and adding a translational perspective.The evolutionarily conserved and ubiquitously expressed SMN protein localizes to both the cytoplasm and the nucleus where it accumulates in nuclear structures known as Gems . The observation that Gems interact with Cajal bodies (CBs), rich in p80/Coilin and RNAs, provided the first indication that SMN functions in RNA metabolism . To thisSMN highly conserved domains include a short segment near the N-terminus that is responsible for binding with high affinity to Gemin2 ; the TudSeveral of these integral components are found in distinct complexes outside of the SMN complex itself  ,25,26,27The SMN complex has been showed to assemble in a stepwise manner through multiple modular additions. These findings also reflect an expansion in the complexity of the SMN complex throughout evolution. Accordingly, the multi-protein human SMN complex arose from an ancestral version in fission yeast comprised of solely SMN and Gemin2 that underwent addition of multiple Gemin proteins . ImportaOn the other side, some of the components of the SMN complex also act independently to modulate the protein synthesis. This is the case of Gemin 5. Gemin 5 plays a crucial role in RNA translation both in synergy with SMN, through binding and delivering RNAs to the SMN complex, as well as outside the SMN complex ,25,26. GIn the following section, we provide a wide overview of the roles played by the SMN complex in RNA processing, describing both the well-established pathways and the most recently described ones .Precursor messenger RNA (pre-mRNA) splicing is a key process during post-transcriptional regulation of gene expression, as it enables the production of a large variety of proteins from a limited pool of genes. Up to 95% of human genes undergo alternative splicing with selective removal of specific introns by spliceosome RNP-dependent machinery . The SMNIn the cytoplasm, the different Sm proteins sequentially associate with each other and with chloride conductance regulatory protein (pICln), which recruits all newly synthesized Sm proteins to the protein arginine methyltransferase 5 (PRMT5) complex ,39. The Cajal body condensation is mediated by different prost-translational modification (PTM) events. These include mTOR-dependent phosphorylation of serine 49 and 63 on SMN, arginine methylation, acetylation, as well as sumoylation ,51,52. TThe U7 snRNP is the only example of a U snRNP that has no function in mRNA splicing but is exclusively dedicated to the 3′-end processing of a select subset of mRNAs that code for the replication-dependent histone proteins ,55. The The SMN complex is required for the assembly of the U7 snRNP  Figure . While UThe functional consequence of U7 snRNP reduction caused by SMN deficiency is a disruption of replication-dependent histone mRNA 3′-end processing resulting in accumulation of aberrantly poly-adenylated transcripts. This indicates that the SMN-dependent reduction in functional U7 snRNP levels does not simply reduce the rate of processing histone transcripts, but switches processing to an entirely different modality of 3′-end processing. The loss of this unique histone mRNA 3′-end structure ultimately leads to dysregulated histone gene expression .Despite the well-characterized role of SMN in the assembly of U snRNPs, there are numerous lines of evidence that situate SMN in the center of a wide variety of RNA processing pathways through interactions with various RNA-binding proteins (RBPs) . The vasInterestingly, SMN also associates with RBPs involved in other aspects of mRNA regulation and with hundreds of transcripts containing AU-rich elements (AREs) in their 3′ UTR  that areR-loops consist of RNA/DNA hybrids, formed during transcription when nascent RNA hybridizes to the DNA template strand, displacing the non-template DNA strand . They weIn line with the fact that R-loops can cause genomic instability, factors implied in DNA damage response are also able to prevent R-loop formation or solve them once formed. They include three topoisomerases  and RNA helicases ,81. In pA recent study investigated the mechanism of methylation of RNAP II carboxy-terminal domain (CTD) , dependeSMA is the leading genetic cause of infant death and the second most common autosomal recessive genetic disorder after cystic fibrosis ,86. SMA The development of various animal models of SMA has been valuable to defining the cellular and molecular basis of the disease ,88. CompExamination of cellular pathology reveals significant loss of motor neurons in the spinal cord of SMA mice ,91,92,93In sum, SMA is emerging as a disease characterized by dysfunction of multiple components of the motor system and not only dysfunctioning motor neurons. Defects in heart, pancreas, and liver have been documented in severe SMA mice ,104,105,In the following section, we provide a broad outline of how dysfunctions in selective SMN-dependent RNA pathways contribute to the SMA phenotype .The proper assembly of the spliceosome machinery relies on the chaperone function provided by the SMN complex. Altered snRNP production due to SMN deficiency can lead to the neuromuscular defects that are hallmarks of the SMA phenotype .Biochemical quantification of snRNP assembly in cellular and animal models of SMA showed a strong correlation between SMN expression levels and disease severity. Accordingly, both fibroblasts and lymphoblasts derived from SMA Type I patients have a reduced capacity for assembly of snRNPs . DeliverImportantly, the reduction in steady-state snRNP levels is not only moderate but also differential. In spinal cords from SMA mice, the components of the U12-dependent spliceosome are more significantly reduced, as compared to other tissues from the same animals ,112,113.While there is little doubt that SMN deficiency causes splicing alterations, the downstream consequences and contribution of this dysfunction to the SMA phenotype is less elucidated. One possibility is that widespread splicing alterations cause the SMA phenotype. This scenario is founded on the identification of widespread splicing defects in tissues from SMA animal models ,116,120.Building on the observations that components of the minor spliceosome are preferentially reduced in the spinal cord of SMA mice and that this pathway may have a prominent role in neuronal homeostasis, a study analyzed all Drosophila minor intron-containing genes to identify those that are not only aberrantly spliced but also lead to functional defects in the Drosophila motor system . SMN defA recent work looked at the specific role of U12 splicing alterations in different SMA mouse models of varying disease severity . The autOther studies have since focused on additional genes that are mis-spliced in SMA models including Neurexin2a (Nrxn2a) and Chondrolectin (Chodl), both of which are important for motor neuron axon outgrowth ,126.These studies demonstrate that SMN-dependent alteration of the snRNP biogenesis pathway can produce specific deficits that, when combined, result in profound motor dysfunction. Continued research is needed to identify the specific molecular mechanisms linking SMN deficiency to downstream target genes alteration and, secondarily, those genes’ relative contributions to the SMA phenotype in a mouse model of the disease.Consistent with the essential role of SMN for U7 snRNP biogenesis, the steady-state levels of U7 snRNP are strongly reduced in both SMN-deficient mammalian cells and tissues of SMA mice . The redDue to increased susceptibility of genotoxic stress in SMA patient-derived fibroblasts, SMN deficiency can reduce expression of histone H2AX and, consequently, decrease levels of its phosphorylated form during DNA damage . The phoA recent study demonstrated that SMN-dependent assembly of U7 snRNP is required for NMJ integrity. The expression of U7-specific Lsm10 and Lsm11 proteins in SMA mice was able to enhance U7 snRNP assembly, rescue histone mRNA processing defects and finally ameliorate the neuromuscular phenotype . InteresAltogether, these findings provided a compelling example of a molecularly defined cascade of events linking SMN deficiency with dysregulation of a select RNA processing pathway through the disruption of a specific RNP assembly activity carried out by SMN.The association of SMN with multiple RBPs suggests it functions to modulate mRNA metabolism beyond nuclear processing by snRNPs. SMN-interacting RBPs are known to bind to a variety of cytoplasmic mRNAs and play a role in multiple regulatory aspects of their life cycle such as stability, transport, and translation. Dysregulation of any of these processes induced by SMN deficiency may contribute to neuronal dysfunction and thus be relevant to disease etiology . Of partThe assembly and transport of mRNPs along the axon is crucial for proper development of the axonal terminals (for review of the role of SMN in this process see ). For exAdditionally, SMN has been reported to bind to HuD, a RBP important for mRNA stability during neural development ,65. The By further identifying SMN’s role in supporting and facilitating axonal development, we can begin to unravel how deficits in this complex contribute to neurodegeneration. Indeed, impaired assembly of mRNPs has been identified as a key mechanism of many neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), in which stress granules containing mRNPs are hallmarks of disease pathology . It seemMutations in proteins that function in R-loop resolution are responsible for devastating—often neurodegenerative—human diseases and indicate that R-loops warrant further understanding in disease conditions . MutatioA recent study proposed that SMN controls R-loop resolution by interacting with the C-terminal domain of RNA polII and SETX  Figure . HoweverTaken together, these findings shed light on the fact that the role played by SMN encompasses a broad spectrum of actions ranging from RNA metabolism to genome stability. However, further investigations will be needed to dissect the molecular pathway linking SMN depletion and R-loop generation and its relevance on the disease phenotype.SMN has also been recently associated with multiple non-coding RNAs metabolism and function . For exaThe SMN locus also produces a wide variety of circRNAs in addition to SMN protein. The mouse Smn locus was projected to produce a significantly lesser amount of circRNAs than the human SMN locus . These vThe progressive dissection of the SMN complex functions in RNA metabolism has led to the development of commercially available RNA-based therapies for SMA, which have drastically changed the outcome of this disease. However, the full picture of the multifaceted functions of SMN in post/transcriptional gene regulation and their effects on downstream cellular pathways that determine the SMA phenotype still need to be fully elucidated.With the present review we showed the multifaced role of the SMN protein and the effect of the loss of discrete functions of SMN in the pathogenesis of SMA . In partA number of interacting proteins are responsible for the achievement of all these roles of SMN, including Gemin 2-8, Unrip, Sm proteins, pICln, snRNAs, hnRNP-R, the RBP HuD, KSRP, and IMP1, β-actin mRNA, as well as 3′ UTR ARE reach transcript , RNAP II CTD and SETX, and non-coding RNAs (including miRNAs and circRNAs) .Mechanistically, it will be important to define how the specificity of interactions among the protein partners and the SMN complex is achieved, and how different post/translational modifications impact on such interactions. These studies will serve as the basis to optimize current therapies, possibly leading to a multi/target treatment. In the near future, restoration of SMN obtained through RNA/based therapeutics could be complemented by SMN/independent approaches aimed at preventing motor neuron death and muscle wasting.


CLN3 and CLN6 to bipolar cells has been shown to attenuate the retinal dysfunction. Therapeutic benefits of ocular enzyme replacement therapies were evaluated in CLN2 and CLN10 animal models. Since brain-targeted gene or enzyme replacement therapies will most likely not attenuate retinal neurodegeneration, there is an unmet need for treatment options additionally targeting the retina in patients with NCL. The long-term benefits of these therapeutic interventions aimed at attenuating retinal degeneration and vision loss in patients with NCL remain to be investigated in future clinical studies.The neuronal ceroid lipofuscinoses (NCLs) are a group of childhood-onset neurodegenerative lysosomal storage disorders mainly affecting the brain and the retina. In the NCLs, disease-causing mutations in 13 different ceroid lipofuscinoses genes (CLN) have been identified. The clinical symptoms include seizures, progressive neurological decline, deterioration of motor and language skills, and dementia resulting in premature death. In addition, the deterioration and loss of vision caused by progressive retinal degeneration is another major hallmark of NCLs. To date, there is no curative therapy for the treatment of retinal degeneration and vision loss in patients with NCL. In this review, the key findings of different experimental approaches in NCL animal models aimed at attenuating progressive retinal degeneration and the decline in retinal function are discussed. Different approaches, including experimental enzyme replacement therapy, gene therapy, cell-based therapy, and immunomodulation therapy were evaluated and showed encouraging therapeutic benefits. Recent experimental ocular gene therapies in NCL animal models with soluble lysosomal enzyme deficiencies and transmembrane protein deficiencies have shown the strong potential of gene-based approaches to treat retinal dystrophies in NCLs. In CLN3 and CLN6 mouse models, an adeno-associated virus (AAV) vector-mediated delivery of  CLN genes are summarized in the NCL mutation database (https://www.ucl.ac.uk/ncl-disease), and genotype-phenotype correlations are discussed in a recent review . The NCLs are caused by defects in 13 different genes  encoding soluble lysosomal proteins , membrane proteins located in the ER CLN6 and CLN8), ER-Golgi intermediate compartment (CLN8) or lysosomes , and cytosolic proteins associated with synaptic vesicles (CLN4) or the plasma membrane CLN14,  (1). The and CLN8t review . With tht review  (10). Hot review . An enzyt review .Cln3ex1−6Δ mice, a CLN3 mouse model  recordings . Intraviype mice . AlthougTPP1-deficient Dachshund model recapitulates the key features of human CLN2 disease, including ataxia, tremor, progressive brain atrophy, loss of vision, and a reduced life span (TPP1 (cTPP1) cDNA into the CLN2 Dachshund model led to the reduced storage of autofluorescent material and decreased astrocytosis in the brain, and delayed onset of cognitive deficits and extended lifespan of the mutant dogs  genetically recapitulates the 1 Kb deletion mutation of exons 7 and 8 found in 85% of human patients with CLN3 disease  Cln6nclf mice reduced the degeneration of photoreceptors in 3-, 6-, and 9-month-old mice compared with untreated controls. In striking contrast, a brain-directed AAV-mediated expression of cathepsin D in a CLN10 mouse model prevented the accumulation of ceroid lipofuscin, the activation of microglia, and neurodegeneration in brain tissues, but not the rapidly progressing retinal degeneration  mice are characterized by an early-onset loss of photoreceptor cells and a subsequent loss of all other retinal nerve cell types, the accumulation of storage material, lysosomal dysfunction, reduced autophagic flux, reactive astrogliosis and microgliosis, and a shortened lifespan with premature death at P26  is a model for the most severe NCL form, congenital NCL. Cathepsin D knockout  mouse, autofluorescent storage material accumulation, and the degeneration of photoreceptors and retinal ganglion cells become apparent in 12-month-old mutant mice  rely on the intravitreal administration of a recombinant soluble lysosomal enzyme and its uptake rrection . Theraperrection .Periodic intravitreal injections of recombinant TPP1 starting at 12 weeks of age into the CLN2 Dachshund dog model led to decreased neurodegeneration in the inner nuclear layer and inhibited declines in ERG amplitudes . Intravivia mannose 6-phosphate receptors by surrounding defective acceptor cells  and transmembrane protein deficiencies (CLN3 and CLN6) have shown the strong potential of gene therapeutic approaches to effectively treat NCL-related retinopathies. A major breakthrough in the experimental gene therapy approaches was the identification of the specific cell types that have to be targeted to achieve therapeutic benefit. In the CLN3 and CLN6 mouse models, the AAV-mediated bipolar cell-specific delivery of CLN3 and CLN6 was successful in preventing the loss of photoreceptors and bipolar cells, respectively, and to partly preserve retinal function. Furthermore, the intravitreal injection of an AAV9-CLN5 vector into a CLN5 sheep model largely prevented retinal degeneration and loss of retinal function. Finally, results from a recent study suggest that an AAV-mediated CTSD expression in the retina of a CLN10 mouse model is more potent in preventing retinal degeneration than intravitreal ERT mediated by the injections of recombinant CTSD or by transplantation of neural stem cells overexpressing CTSD. While the results of the eye-targeted therapies are encouraging, most studies have started the treatment prior to the onset of the retinal pathology. Future work thus needs to evaluate whether the treatment strategies are still effective when they are started at the initial or advanced stages of the retinal dystrophy. For clinical applications, gene therapies have the advantage of single dosing compared with enzyme replacement therapies which require repeated administration. Combined therapies targeting the brain and the retina separately may attenuate neurological symptoms and additionally vision loss in patients with NCL. The long-term benefits of these experimental ocular treatment options have to be evaluated in patients with NCL in future clinical studies.Retinal degeneration and loss of vision are among the major hallmarks of NCLs of NCLs. With the exception of one study on UB and SS interpreted the data and wrote the review. Both authors contributed to the article and approved the submitted version.The laboratory works of the authors were supported by funding from the Mila's Miracle Foundation (SS), the Stiftung zur Förderung der Universitätsmedizin Hamburg, and the Ernst and Berta Grimmke Stiftung (UB).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.


Osteoarthritis (OA) is a degenerative disease characterized by loss of articular cartilage and chronic inflammation, involving multiple cellular dysfunctions and tissue lesions. The non-vascular environment and dense cartilage matrix in the joints tend to block drug penetration, resulting in low drug bioavailability. There is a desire to develop safer and more effective OA therapies to meet the challenges of an aging world population in the future. Biomaterials have achieved satisfactory results in improving drug targeting, prolonging the duration of action, and achieving precision therapy. This article reviews the current basic understanding of the pathological mechanisms and clinical treatment dilemmas of OA, summarizes and discusses the advances for different kinds of targeted and responsive biomaterials in OA, seeking to provide new perspectives for the treatment of OA. Subsequently, limitations and challenges in clinical translation and biosafety are analyzed to guide the development of future therapeutic strategies for OA. As the need for precision medicine rises over time, emerging multifunctional biomaterials based on tissue targeting and controlled release will become an irreplaceable part of OA management. Osteoarthritis (OA) is one of the most common chronic degenerative joint diseases worldwide, often occurring in middle-aged and older adults over the age of 45 In recent times, many biomaterial carriers with cartilage or synovial targeting and responsiveness are emerging. Biomaterial carriers not only prolong the release of drug, but also exhibit higher tissue and cell specificity In this review, we briefly overview the underlying physiopathology of OA and the currently used clinical treatments and their dilemmas. Then, we describe and summarize the characteristics of different types of targeted and responsive biomaterials and their research progress in OA. Finally, we discuss the potential limitations and challenges of biomaterials in the translation of clinical treatments for OA. We hope to provide further references and new perspectives for the future development of targeted and responsive biomaterials in OA therapy.The anatomy of articular cartilage is divided into hyaline cartilage, tidemark, and calcified cartilage According to the causative factors, OA can be broadly divided into primary OA caused by gene, age and gender factors, and secondary OA caused by sports trauma, metabolic disorders, obesity, etc. At the molecular level, Interleukin-1β (IL-1β), produced by chondrocytes, osteoblasts, synoviocytes and macrophages, is considered to be the most critical inflammatory factor in the pathogenesis of OA, which can act independently or synergistically with other cytokines to cause articular cartilage degradation and joint inflammatory responses The clinical treatment of OA is divided into two main categories: medication and surgery, while medication is split into two groups: symptom relief and condition improvement in vitro culture techniques, autologous chondrocyte implantation (ACI) is better able to regenerate normal articular cartilage structures In advanced OA, severe joint deformities will occur, rendering pharmacological treatment futile, and surgical treatment is the only option, most commonly with arthroscopic debridement and total joint replacement Currently available anti-inflammatory and analgesic drugs, either orally or by intra-articular injection, suffer from poor water solubility, low cellular uptake, poor drug concentration distribution or premature degradation Since enzymes possess a high degree of selectivity and efficiency, drug release in a variety of ways such as surface ligand activation and chemical bond breaking can be achieved by introducing functional groups sensitive to enzymes aberrantly expressed in the OA microenvironment into biomaterials Although enzymes have received extensive attention in studies on their application as triggers in malignancies and cardiovascular diseases, their application in OA therapy is still in its infancy so far 2-), hydrogen peroxide (H2O2), hydroxyl radicals (OH·), nitric oxide radicals (NO·) and singlet oxygen (1O2), which have high chemical reactivity because they contain unpaired electrons ROS broadly refers to oxygen-derived radicals and non-radicals, and contains superoxide anions (O· (1000 mM), and all thioketal bonds were broken, leading to nanoparticle depolymerization and structural relaxation In vivo experiments, PTKU NPs not only demonstrated favorable articular targeting, but also boosted the shift of synovial macrophages to M2 type and downregulate the expression of MMP-2 and various inflammatory factors in articular cartilage in vitro catalysis of KO2, TKCP@Dex NPs progressively broke down the thioketone bond to release Cy5.5 and Dex to visualize the treatment, scavenging IL-6 and MMP-13 without causing cytotoxicity The mechanisms of ROS-responsive biomaterials are divided into two types: chemical bond breaking  and hydrophilic-hydrophobic conversion  2O2 passes through the PLAG shell and then oxidizes ethanol to acetic acid by the Fenton reaction with Fe2+ catalysis, subsequently sodium bicarbonate decomposes in an acidic environment to produce abundant carbon dioxide (CO2), which eventually leads to the collapse of HM 2O2/pH 6.8, making this unique structure ideal for the release of cargo Compared with the previous study, the water-in-oil-in-water biphasic hollow microspheres (HM) composed of ethanol, ferrous chloride, Dex, and sodium bicarbonate achieved ultra-sensitive ROS responsiveness as shown in Figure In OA lesioned joints, synovial fluid pH decreases from 7.4 to 6.0 owing to local tissue hypoxia metabolism and acidosis + is present at high levels, the protonation of free NH2 in Cs leads to increased water solubility and rupture of the protective layer, triggering pH-responsive cargo release 2 with NH4HCO3 to lead to lactate disintegration, then Rh-PLGA-NPs@NH4 released three times as much rhein (Rh) as the control in the OA synovial fluid mimic Additionally, some strategies are based on pH-dependent hydrophobic alterations or internal gas generation, allowing for overall morphological structural changes to achieve on-demand drug release In contrast to endogenous factors like enzymes, ROS, and pH, simple and manipulable exogenous stimuli have also been used to initiate the release of loaded drugs in vitro, but also rescue cartilage matrix deficiency and abnormal ossification  in vivoAfter the onset of inflammation, clinical practice reveals swelling of the knee joint accompanied by an increase in temperature of 2-3°C in vivo MRI, enabling the integration of diagnosis and treatment Magnetic manipulation has attracted interest due to its biocompatibility and non-invasive operation 2O2 nanoenzymes produce O2 to alleviate hypoxia in the joint cavity, thereby downregulating hypoxia-inducible factors to prevent angiogenesis Ultrasound-responsive biomaterials for arthritis treatment are being developed in recent years. Liao et al. successfully delivered diclofenac sodium to rat joints by ultrasound responsiveness In the treatment of OA, whether by intra-articular injection or systemic injection, the disorganized diffusion of the drug agent in the organism is fundamental to the efficacy of the drug 2 nanoparticles by chondrocytes was observed at 30 μm after 24 h incubation with bovine cartilage explants As an avascular dense connective tissue, the cross-linked network formed by type II collagen with a pore size of about 50-60 nm and aggrecan with a chain spacing of about 20 nm hinders the uptake of most drugs In addition, the constituent monomers of Aggrecan contain a large number of negatively charged carboxyl residues and sulfate residues, allowing cationic biomaterials to be attracted to the joints by electrostatic forces in vivo studies in different model animals in vivoin vitro live imaging in vivoThe identification of highly specific targeting ligands is often the paving stone for the study of active targeting strategies. In 2011 cartilage-targeting peptides  screened by phage display technology have been shown to specifically induce the binding of nanoparticles to chondrocytes in vivo imaging by accumulating at the injured joint after intravenous injection in vivo protection against ectopic calcification of synovial membrane, which was superior to steroid treatment To further improve the active targeting of vectors, specific antibodies against articular cartilage have been incorporated into biomaterial fields of application. Compared with other methods, the antibody only binds and tightly attaches to the antigenic epitope on the surface of the corresponding antigen with high affinity and specificity, greatly improving the targeting of the vector in vivo and in vitro studies to clarify the action mechanisms and long-lasting therapeutic effects, restricting their clinical translation. Overall, active targeting strategies based on cartilage and extracellular matrix are still in their infancy, further development of more desirable OA-targeting biomaterials can be achieved in the future by drawing on experience from other diseases such as cancer or immunomodulation.For biocompatibility and adaptability considerations, some bionic biomaterials have also been applied for active targeting of OA + (VWF+) vascular endothelial cells and CD34+ hematopoietic stem cells in synovial tissue Synovium is composed of macrophages, fibroblasts and vascular endothelial cells, which perform the functions of material exchange and secretion of synovial fluid 2O2 in activated macrophages while decreasing mitochondrial membrane potential and ATP synthesis, yet showed a satisfactory safety profile for normal macrophages in vivo as a promising therapeutic strategy for OA.In the inflammatory microenvironment, interferon-γ (IFN-γ) and TNF-α induce synovial macrophages to polarize toward the pro-inflammatory M1 type, leading to the release of downstream inflammatory factors IL-1β and IL-6 Although cartilage degeneration and ECM loss have long been considered the primary causative factors of OA, there is growing evidence that subchondral bone lesions precede the onset of cartilage loss in vivo evaluation of simulated biomaterials using in vitro stem cell models or bone like organs has recently received much attention, which would be more convincing than toxicity experiments with single cell lines Although biomaterials hold exciting promise for improving drug solubility and precision release, the potential risks and unknown chronic toxicity of these materials in living organisms limit their clinical translation. The size of most biomaterials reaches the nanometer dimension, which allows them to easily cross multiple biological barriers and be non-specifically endocytosed by immune cells The biodistribution and toxicity of the delivery platforms within the joint will also be influenced by the strength of the surface charge. Positively charged cationic materials tend to have easier uptake by cells and are therefore often used for therapeutic RNA delivery Due to the presence of synovial fluid, adsorption of synovial proteins on the surface of biomaterials will probably interfere responsive group breakage and exposure of targeting ligands Finally, researchers should be cautious in their choice of model animals and means of inducing OA. Due to differences in species, age, and sex, none of the animal models can fully replicate all pathological processes in human OA OA is a degenerative disease characterized by loss of articular cartilage and local inflammation that affects tens of millions of people worldwide. The unknown pathogenesis of OA leads to clinical treatment strategies that do not completely eradicate cartilage degradation. Conventional intra-articular injections or oral administration of drugs are often rapidly cleared by phagocytes or metabolic organs resulting in low drug utilization and a narrow therapeutic window. The application of targeted and responsive biomaterials in the biomedical field is bringing new light to the treatment of OA. Efficient drug delivery systems based on the OA microenvironment  and lesion sites  have shown promising applications. Not only do they maximize the therapeutic potential of drug molecules, but more importantly they improve the biodistribution and delivery of drugs. The "bio-missile" based on biomaterials penetrates nanoscale pores and has long-lasting controlled release, avoiding the pain and economic burden of multiple high-dose administrations to patients. By modifying and optimizing the structure and physicochemical properties, biomaterials overcome the therapeutic bottleneck of drug monomers and enable the integration of treatment. The controlled release of drugs in combination with microenvironmental changes assists in better mastering the pathological stages of OA, while the coupling of targeted ligands opens new horizons for precision therapy. Moreover, these targeted and responsive biomaterials may be used for other OA-affected joint tissues in the future, including fat pads and joint ligaments.Nevertheless, biomaterial-based clinical therapies for OA are still in their infancy, and there is a lack of sufficient clinical studies to demonstrate the efficacy and safety of these strategies. Previous studies have also shown that the selection of natural cartilage components based on hyaluronic acid and collagen as carriers may yield better therapeutic benefits and safety. Also, the cartilage layer is thicker in primates, which would greatly prolong the diffusion of biomaterials. These findings should be further validated in a more closely related human model . In addition, further validation is needed to determine if the effectiveness of these targeted ECM strategies is compromised in the late stages of OA when the collagen network of chondrocytes is depleted. On the other hand, new multifunctional biomaterials involve more complex and sophisticated technologies, often leading to expensive economic costs in clinical applications, which should be taken into account. Since the criteria for toxicity evaluation of biomaterials at the development stage are not yet standardized, the absence of a quality evaluation system will lengthen the cycle of the approval process, which is not conducive to the clinical translation of biomaterials. To break the existing barriers and accelerate the clinical translation of targeted and responsive biomaterials, we need to deepen our understanding of OA not only at the molecular and cellular levels, but also to speed up the research process of novel active drug molecules and the search for more specific drug carriers to achieve stronger delivery effects. To meet these challenges, broad and close collaboration and cross-disciplinary research among materials scientists, biologists and clinicians are required. Looking ahead, with the continuous improvement of regulatory mechanisms and experimental techniques, the targeted and responsive biomaterials are expected to become a panacea for OA patients.


Plants utilize delicate mechanisms to effectively respond to changes in the availability of nutrients such as iron. The responses to iron status involve controlling gene expression at multiple levels. The regulation of iron deficiency response by a network of transcriptional regulators has been extensively studied and recent research has shed light on post-translational control of iron homeostasis. Although not as considerably investigated, an increasing number of studies suggest that histone modification and DNA methylation play critical roles during iron deficiency and contribute to fine-tuning iron homeostasis in plants. This review will focus on the current understanding of chromatin-based regulation on iron homeostasis in plants highlighting recent studies in Arabidopsis and rice. Understanding iron homeostasis in plants is vital, as it is not only relevant to fundamental biological questions, but also to agriculture, biofortification, and human health. A comprehensive overview of the effect and mechanism of chromatin-based regulation in response to iron status will ultimately provide critical insights in elucidating the complexities of iron homeostasis and contribute to improving iron nutrition in plants. Multiple studies have also revealed that changes in chromatin state by histone modification or DNA methylation play important roles in nutrient homeostasis in plants  . Despitevia a reduction-based process known as Strategy I, which involves proton efflux to the rhizosphere by proton ATPases such as AHA2 to solubilize ferric chelates  family transporters  expression via H3K4me3 , AtFER3, and AtFER4 in iron sufficient seedlings  is typically associated with gene repression; it spreads along the chromatin, resulting in compaction and the silencing of targeted genes . H3K27meeir loci . Howevereir loci . Transcrclf mutants accumulated less iron in the roots than the shoots, but clf seedlings still had higher levels of iron compared to wild type. This phenotype and the higher expression of iron acquisition genes in clf roots  catalyzes the symmetric dimethylation of histone4 arginine3 (H4R3sme2) and regulates diverse biological processes including response to salt stress . SKB1-me to iron . While At on FIT . The mecHistone acetylation is generally associated with transcriptional activation, in contrast to the more complex effects of histone methylation on gene expression . The comFERRIC REDUCTASE DETECTIVE3 (AtFRD3), which encodes a transporter that loads citrate into the xylem to aid translocation of iron-citrate complexes to the shoots , a DNA methylation inhibitor, and the loss of OsDRM2, a key methyltransferase responsible for CHH methylation, resulted in lower expression of OsIRO2 and OsbHLH156, accumulation of less iron, and growth retardment under iron deficiency  and exhibited increased global DNA methylation that resembled the methylation profile of rdd triple mutants (rdd mutants were more tolerant against cadmium stress and accumulated more iron in the shoots by expressing higher levels of iron deficiency response genes than wild type. However, inadequate iron supply abolished cadmium tolerance in rdd mutants (DNA methylation status was also proposed to be involved in feedback mechanisms between iron status and tolerance to cadmium stress . Arabido mutants . The rdd mutants .via histone modification or DNA methylation. Global changes in H3K9me2 and H3K4me3 levels under high iron stress conditions were detected in the proximal root meristem in rice (Increasing evidence has shown that iron homeostasis gene expression is affected by histone modification  and DNA  in rice , but fur in rice . Thus, tJS primarily wrote the initial draft of the manuscript. ZY and YW contributed to the manuscript writing and generated the figures. JL and JJ conceived the idea and made final edits. All authors contributed to the article and approved the submitted version.


Recently, a growing body of evidence has indicated that secondary neurodegeneration after stroke occurs at remote regions of the brain that are connected to the primary infarction site. For instance, following a middle cerebral artery stroke, secondary neurodegeneration was observed in the ipsilateral thalamus, substantia nigra, white matter tract and hippocampus, despite these regions being remote from the site of primary infarction ,2. The nAccumulated data indicate that neuroinflammation, as demonstrated by the activation of resident inflammatory cells such as microglia and astrocytes, as well as infiltrating peripheral inflammatory cells, is a prominent hallmark of secondary neurodegeneration in pre-clinical and human studies ,8,9. Tur12 receptor, P2Y6 receptor and complement receptor 3, were discussed as potential targets to reduce secondary neurodegeneration after stroke. In a subsequent communication paper, Brown and Milde investigated the role of the microglial phagocytic P2Y6 receptor in the prevention of neuronal loss after stroke [6 receptor knockout mice, they demonstrated that neurons were spared from microglial phagocytosis, suggesting that inhibition of the P2Y6 receptor after stroke may be beneficial to prevent delayed neuronal loss by microglial phagocytosis. Microglia, the primary resident inflammatory cells of the CNS, play an important role in responding to stroke-induced tissue injury within the primary infarction site as well as the sites of secondary neurodegeneration ,11. Browr stroke . Using PStress is known to negatively interfere with rehabilitation and is associated with poorer functional outcomes post-stroke . The hypThe enhancement of brain recovery after stroke remains a challenging area, with no therapeutic intervention being widely adopted to promote the recovery of brain function and improve functional outcomes in clinical practice. Clarkson and team investigated the therapeutic potential of glycomimetic compounds along with recombinant human recombinant human brain-derived neurotrophic factor in alleviating post-stroke cognitive impairment . They deTaken together, this Special Issue offers critical insights to the mechanisms related to secondary neurodegeneration after stroke and suggests potential strategies that may help to improve recovery after stroke. Further research in this space is needed to better understand the pathophysiology of secondary neurodegeneration after stroke, and more rigorous pre-clinical studies and human trials should be conducted to validate the potential therapies.


Stroke is the second leading cause of death worldwide and the vast majority can be attributed to modifiable risk factors, mainly behavioral and metabolic. The top six risk factors responsible for cardiovascular mortality in Brazil in 2019 were high systolic blood pressure, inadequate dietary exposure, high body mass index, high LDL cholesterol, high fasting blood glucose levels, and tobacco. We intend to discuss in this paper the evidence and recommendations in the approach of three essential risk factors for patients with a history of stroke: dyslipidemia, hypertension and diabetes.  Stroke is the second leading cause of death worldwide and the third leading cause of disabilityIn recent years, studies have shown a reduction in recurrent stroke and transient ischemic attacks (TIAs) rates, as prevention strategies have been implemented and improvedThe Global Burden of Disease (GBD) Study showed that approximately 90% of strokes could be attributed to modifiable risk factors, including behavioral factors such as smoking, poor diet, sedentary lifestyle, and metabolic factors such as high blood pressure, blood glucose, and cholesterolAlthough the combination of dietary modification, physical activity, use of aspirin, statins, and antihypertensive agents may result in a cumulative reduction in the relative risk of stroke recurrence by 80%Elevated low-density lipoprotein cholesterol (LDL-c) levels are associated with an increased risk of ischemic stroke, but this correlation is not evident in patients with hemorrhagic stroke. In addition, the incidence of atherothrombotic and lacunar strokes, but not of cardioembolic strokes, increases significantly with increased LDL-c levelsGiven the association between serum LDL-c levels and ischemic stroke, the SPARCL (The Stroke Prevention by Aggressive Reduction in Cholesterol Levels[A1]) study compared the use of high-potency statins with placebo in patients who had had a stroke or TIA in the previous six months and had no known coronary disease. A 16% relative risk reduction for stroke was observed in the atorvastatin group, which is partly explained by the lower mean LDL-c level achieved in this group compared to placebo . However, the anti-inflammatory effect of statins and reduction of endothelial dysfunction also contribute to this findingDue to the residual risk of cardiovascular events despite high-potency statins therapy, additional treatments to reduce LDL-c have been implemented. More recently, the IMPROVE-IT study  evaluated the use of Ezetimibe, a cholesterol absorption inhibitor, associated with statins in patients hospitalized for acute coronary syndrome, showing a significant reduction in the risk of ischemic stroke when compared with the use of statins alone, during a mean follow-up of six years,Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, a class of monoclonal antibodies represented by evolocumab and alirocumab, are capable of reducing serum LDL-c levels by approximately 60%, proving to be a promising therapeutic class. The FOURIER study (Further Cardiovascular Outcomes Research with PCSK9 Inhibition in Subjects with Elevated Risk) evaluated the use of evolocumab versus placebo in patients with established atherosclerotic disease and LDL-c levels above 70 mg/dL in spite of statin therapy. The addition of evolocumab reduced LDL-c levels from a median of 92 mg/dL (2.4 mmol/L) to 30 mg/dL (0.78 mmol/L), with a consequent reduction in the rate of ischemic strokes, but no significant difference concerning hemorrhagic strokes. This finding was consistent in the subgroup of patients with previous ischemic stroke and those without a history of strokeA meta-analysis of randomized controlled trials evaluated the risks and benefits of a more intensive reduction in LDL-c when compared to more permissive therapy. The final mean LDL-c level in the former group, adjusted for the size of the study, was 79 mg/dL, compared with 119 mg/dL in the latter group. More intensive treatment was associated with a decrease in the risk of recurrent stroke. The benefit was not statistically different between the various LDL-c reduction strategies, demonstrating that the serum cholesterol level reached is more important than the therapy itselfCurrent guidelines recommend that, in the presence of clinically manifested atherosclerotic disease in patients with previous ischemic stroke, LDL-c reductions of at least 50% should be considered. It is ideal to obtain LDL-c levels < 50 mg/dL. For primary prevention, LDL-c levels < 70 mg/dL are recommended in high-risk patients, < 100 mg/dL in those at intermediate risk, and < 130 mg/dL in those at low riskLipoprotein(a) [Lp(a)] is a plasma lipoprotein consisting of a cholesterol-rich LDL particle with an apolipoprotein B100 molecule attached to an additional protein, apolipoprotein A (ApoA). It is synthesized by the liver, and more than 90% of its circulating levels are genetically determined, with little influence of diet or environmental factorsSeveral studies have suggested that high serum Lp(a) values constitute an independent risk factor for atherosclerotic disease through multiple distinct mechanisms. Lp(a) can be deposited on the vessel walls, and due to its LDL and ApoA particles' high propensity to oxidation, these particles become highly immunogenic and proinflammatory. The fact that Lp(a) is more subject to oxidation than LDL can facilitate its uptake by macrophages, which become foam cells, atherosclerosis precursors. In addition, ApoA is structurally similar to plasminogen, interfering with the fibrinolytic cascade and resulting in a high risk of thrombogenicityAlthough the role of Lp(a) as a risk factor for stroke is not as well documented as for coronary artery disease, a meta-analysis of 41 studies showed an increased risk of ischemic stroke and large-artery atherosclerosis in individuals with elevated serum Lp(a) levelsThe use of statins seems to be associated with an increase in Lp(a). Thus, although statin therapy results in cardiovascular protection, it is possible that patients who develop an increase in Lp(a) after starting statin therapy do not obtain the full benefitWhile randomized trials evaluating therapies that reduce Lp(a) by 20 to 30%, such as niacin and cholesteryl ester transfer protein (CETP) inhibitors, did not provide evidence that Lp(a) reduction decreases the risk of cardiovascular events, recent data with PCSK9 inhibitors suggested a possible role in this regardA post hoc analysis of the ODYSSEY Outcomes  study evaluated the benefit of associating a PCSK9 inhibitor with optimized statin treatment in patients with LDL-c close to 70 mg/dL. The effects were evaluated according to the concomitant Lp(a) levels, and individuals with serum levels < 13.7 mg/dL had no reduction in cardiovascular events with the use of alirocumab, unlike those with at least slightly elevated levelsAntisense oligonucleotides such as mipomersen and pilacarsen are small nucleotide sequences (DNA or RNA) that bind specifically to messenger RNA and inhibit protein synthesis. These molecules inhibit the hepatic synthesis of apolipoprotein B100, consequently reducing Lp(a) plasma concentrations. A randomized study evaluated their effect on Lp(a) in patients with established cardiovascular disease, demonstrating a dose-dependent decrease in Lp(a) serum levels, with a mean percentage reduction ranging from 35% to 80%Lp(a) dosage should be considered at least once in a lifetime for every adult. The assessment of Lp(a) plasma levels may also be helpful in patients with a family history of premature cardiovascular disease and determine treatment strategies in individuals with a borderline estimated risk in the risk categoriesSystemic arterial hypertension (SAH) is widely known to be the leading risk factor for stroke, and up to 50% of stroke events can be attributed to SAH. Clinical trials have documented that blood pressure control reduces stroke mortality in hypertensive patients and its recurrence in patients with a history of stroke or TIA. Thus, blood pressure management has become a primary and secondary prevention cornerstone.There is robust evidence that SAH screening and treatment prevents cardiovascular disease and reduces mortalityThe SPRINT study  demonstrated a reduction in cardiovascular outcomes in high-risk hypertensive individuals submitted to more stringent pressure control (target systolic blood pressure [SBP] < 120 mmHg) when compared to more permissive treatment (target SBP < 140 mmHg)For hypertensive patients over 65 years of age, the current recommendation is to maintain blood pressure below 140/90 mmHg. Nevertheless, the recently published STEP (Strategy of Blood Pressure Intervention in Elderly Hypertensive Patients) study, which involved hypertensive patients aged 60 to 80 years, showed a lower incidence of cardiovascular events, including stroke, in those undergoing intensive treatment (SBP target 110-130 mmHg) compared to standard treatment (SBP target 130-150 mmHg)There are gaps in the evidence for blood pressure management in the secondary prevention of stroke, and more evidence is needed. The BOSS study  evaluated the association between systolic blood pressure and clinical outcomes in patients with a history of ischemic stroke or TIA. It demonstrated a probable "J"-shaped curve effect, in which both SBP < 115 mmHg and ≥ 165 mmHg were related to more significant recurrence of stroke when compared to SBP values between 125 and 134 mmHgA meta-analysis of four randomized controlled trials comparing the effect of standard versus intensive blood pressure control in stroke recurrence showed more significant benefit in the latter groupAdditional studies are needed to determine the optimal moment for lowering blood pressure after stroke. Therefore, such recommendations concern the outpatient treatment of patients with stable neurological status.Type 2 diabetes mellitus (T2DM) is a significant risk factor for macrovascular complications, including acute myocardial infarction and stroke, which account for approximately 80% of deaths in T2DM patientsThe contemporary concept of diabetes treatment for patients with established, or those at high risk of, heart disease involves not only glycemic control but also the use of drugs with proven reduction in cardiovascular events. There is now robust evidence for two new classes of drugs: the sodium-glucose cotransporter 2 (SGLT2) inhibitors and the Glucagon-like peptide 1 (GLP-1) analogs. Their evidence in major cardiovascular events reduction is greater than evidence favoring metforminSodium-glucose cotransporter 2 (SGLT2) inhibitors are a new class of oral hypoglycemic agents for the treatment of T2DM, their mechanism being the inhibition of glucose and sodium reabsorption from the proximal renal tubule, thereby reducing glucose levels in the blood. A meta-analysis involving five randomized controlled trials showed no significant effect of this drug class on stroke rates. Subgroup analysis indicated that SGLT2 inhibitors were not associated with fatal and non-fatal stroke, ischemic stroke, and TIA reductions. However, they were associated with a significant reduction in hemorrhagic stroke compared to placeboGlucagon-like peptide 1 (GLP-1) analogs are another class of oral hypoglycemic agents whose mechanism lies in increasing insulin secretion and reducing glucagon secretion in a glucose-dependent manner. A recent meta-analysis demonstrated a reduction in stroke with their use, and additional analyses also demonstrate a reduction in cardiovascular events in patients with a history of previous strokeTherefore, for diabetic patients with cardiovascular disease, namely previous ischemic stroke due to atherosclerosis, regardless of previous metformin therapy, there is an indication for prescription of GLP-1 analog or SGLT2 inhibitors. After the initial therapy, the goal of hemoglobin A1c (HbA1c) must be assessed and additional therapy prescribed accordingly,The therapeutic approach of diabetes in patients with a previous episode of stroke or transient ischemic attack should be individualized, as literature still lacks good evidence regarding goals of HbA1c in this specific population. According to a couple of relatively old studies that compared intensive versus lenient approaches in a general population of diabetes, an aggressive strategy delays the progression of microvascular complications related to diabetesIn conclusion, the assertive approach of risk factors for atherosclerosis is fundamental for the primary and secondary prevention of stroke . Non-pha


Left ventricular diastolic dysfunction is a major cause of heart failure and carries a poor prognosis. Assessment of left ventricular diastolic function however remains challenging for both echocardiography and conventional phase contrast cardiac magnetic resonance. Amongst other limitations, both are restricted to measuring velocity in a single direction or plane, thereby compromising their ability to capture complex diastolic hemodynamics in health and disease. Time-resolved three-dimensional phase contrast cardiac magnetic resonance imaging with three-directional velocity encoding known as ‘4D flow CMR’ is an emerging technology which allows retrospective measurement of velocity and by extension flow at any point in the acquired 3D data volume. With 4D flow CMR, complex aspects of blood flow and ventricular function can be studied throughout the cardiac cycle. 4D flow CMR can facilitate the visualization of functional blood flow components and flow vortices as well as the quantification of novel hemodynamic and functional parameters such as kinetic energy, relative pressure, energy loss and vorticity. In this review, we examine key concepts and novel markers of diastolic function obtained by flow pattern analysis using 4D flow CMR. We consolidate the existing evidence base to highlight the strengths and limitations of 4D flow CMR techniques in the surveillance and diagnosis of left ventricular diastolic dysfunction. Left ventricular diastolic dysfunction (LVDD) is characterized by increased ventricular stiffening coupled with impaired myocardial relaxation. Although these processes occur naturally with aging, they are accelerated and worsened in many cardiovascular conditions.Left ventricular diastolic dysfunction is a major cause of heart failure syndrome in the form of heart failure with preserved ejection fraction (HFpEF), which carries a poor prognosis , 2. Due The diagnosis of HFpEF however remains challenging because of a lack of consensus on the assessment of left ventricular (LV) diastolic function, and because our understanding of complex diastolic hemodynamics is incomplete. Furthermore, many patients are incorrectly diagnosed because the symptoms of heart failure may overlap with manifestations of other conditions . As a result, there is high need for good imaging assessment of LVDD, to provide a positive reason for diagnosing this condition, and avoid overreliance on symptoms to make the diagnosis of HFpEF.In this review, we examine a potentially useful diagnostic tool in diastolic heart failure—diastolic left ventricular blood flow assessment with four-dimensional flow cardiac magnetic resonance (4D flow CMR). This is an emerging technology which has the potential not only to improve early detection of LVDD, but also to expand our understanding of diastolic hemodynamics and ventricular function by going beyond the conventional parameters of LV diastolic function.Echocardiography has traditionally been the modality of choice for non-invasive assessment of LV diastolic function. Current American Society of Echocardiography (ASE) and European Association of Cardiovascular imaging (EACVI) guidelines  recommenCardiac magnetic resonance (CMR) is recognized as the gold standard modality for measurement of cardiac chamber dimensions, volumes, and systolic function, but it can also be used to assess LV diastolic function. Two-dimensional phase contrast CMR (referred to as “2D PC-CMR” for the purpose of this review) measurements of trans-mitral, mitral annular and pulmonary venous velocities, as well as flow, enable diagnosis and assessment of LVDD . DiastolFour-dimensional flow cardiac MRI (4D flow CMR) involves the acquisition of time-resolved three-dimensional phase contrast imaging with three-directional velocity encoding , 14. WitUsing 4D flow CMR, the velocity vector is measured everywhere in a 3D volume over the cardiac cycle allowing the retrospective creation of velocity maps, and by extension flow, at any point in the acquired 3D data volume , 15. 4D In the past decade with greater experience and advances in acquisition techniques, post processing and analysis, 4D flow CMR has become more feasible and clinically relevant as summarized by Crandon et al.  and DemiIn terms of assessment of LV diastolic function, 4D flow techniques such as retrospective valve tracking, can be used to accurately measure conventional flow parameters of LV diastolic function such as mitral inflow velocities with good correlation with echocardiographic measurements , 23, 24.Many different flow pattern-based parameters have been explored by 4D flow CMR studies for more in-depth evaluation of LV diastolic flow hemodynamics and diastolic function . AdditioAs the LV actively expands in diastole and ‘sucks’ blood in from the left atrium, it confers kinetic energy and pressure to the blood. Based on Newton’s second Law of Motion, blood flow kinetic energy (KE) can be calculated using the equation:3) × voxel volume). or stroke volume (SV) to negate the influence of heart size.The conservation or loss of kinetic energy during diastole (diastolic KE) is thought to be a more reliable and direct marker of diastolic work than other parameters such as mitral inflow and myocardial velocity . MoreoveIn normal hearts there are 3 KE peaks , 31, 32,Steding-Ehrenborg et al.  found thSeveral studies have assessed the effects of age on diastolic KE. Early diastolic peak KE declines with age , 30 Fig with valSeveral studies have also assessed diastolic KE in heart failure patients  30, 31,,31, 34. Only one study has examined diastolic KE in the context of valvular heart disease. Al-Wakeel et al.  comparedNot all the energy from myocardial relaxation is transferred into blood flow kinetic energy. Viscous Energy Loss (EL) represents the energy lost to heat , and Turbulent Kinetic Energy (TKE) refers to energy dissipated into small turbulent eddies. Both parameters are novel markers of flow inefficiency but have so far mostly been used to study energy loss in the context of congenital heart disease , 38 and Energy loss has been examined in a single study focusing on LV diastolic function  Table 2Table 2. Zajac et al.  comparedvia this method are not reliant on correct identification of the direction/plane of maximum flow velocity and provide additional 3D spatial and temporal information. These relative pressure maps also present an attractive non-invasive alternative to catheter-based pressure measurements, which are susceptible to operator variability particularly with regards to catheter positioning. However, despite its promising potential as a non-invasive means of measuring intracardiac pressure derangements seen in diastolic dysfunction, relative pressure has yet to be studied in this condition or indeed compared against catheter measurements. Furthermore, current sequences add significantly to scanning time, and there is still much research in progress to refine the computational approach used to derive relative pressure maps from 3D flow velocity data  can also be derived from intraventricular pressure gradients , 49. ChaAs the left atrium empties into the LV and blood passes through the distal tips of the mitral valve, two ring-shaped  vortices are formed during early and late diastole . AlthougVortex assessment using 4D flow CMR is a relatively recent development, echocardiography studies have previously characterized vortex formation and have demonstrated that vortex malformation is associated with energy loss and diastolic dysfunction , 59. EarIn normal physiology, the early diastolic vortex ring is smaller, more circular, and pulls blood toward the apex before dissipating at diastasis. A subsequent larger late diastolic vortex ring then forms which helps direct blood toward the LVOT , 62, 63.A substantial proportion of the kinetic energy of diastolic blood flow is carried within the vortex, with Steding-Ehrenborg et al.  suggestiVorticity is a measure of the local rotation of fluid particles within a fluid as they travel through its main flow. Greater vorticity during ventricular inflow—which is dominated by a large ring vortex—is associated with conservation of kinetic energy leading to more efficient flow . This roinflow. Outflow consists of DF and DEF, the two components that leave the ventricle during systole. RIF and RV remain in the ventricle during systole, making up the Non-ejected Volume.First described by Bolger et al.  using paIn normal hearts, DF is the largest component by proportion of LVEDV (∼35–40%), followed by RV (∼25–30%), with the remaining volume shared equally between RIF and DEF .The relative volume of the flow components reflects flow distribution within the ventricle and can be used as a measure of blood flow efficiency, with reduced relative proportions of DF and RV corresponding to a reduction in systolic but also diastolic function. This is because diastole is integral to creating favorable conditions for maximum DF and thereby flow efficiency. By the end of diastole, DF retains the greatest amount of kinetic energy of all the flow components  and is oSeveral studies have shown alterations in flow component relative volumes between patient groups and under certain conditions .In three different studies, Eriksson et al. , 72, 74 Another study which points to the sensitivity of blood flow component analysis is Karlsson et al. . In thisSundin et al.  studied Alongside assessing flow component volumes, 4D flow CMR techniques can also be used to measure the kinetic energy of individual components. The sum of the diastolic KE of the four flow components are equal to LV global flow diastolic KE. Several studies have shown a direct correlation between the flow component volume and flow component diastolic KE. This is unsurprising given that mass (volume) is a central component of KE. Consequently, component KE normalized to volume  has been reported by many as this provides incremental value .Studies of component diastolic KE have found that an increase in Direct Flow proportion was accompanied by an increase in DF end diastolic KE and a reduction in that of the residual volume end diastolic KE , 78, andLastly, the impact of dyssynchrony on flow component diastolic KE has also been studied. Zajac et al.  comparedFew studies have compared novel 4D flow diastolic parameters against conventional echocardiographic indices of LV diastolic function—Doppler transmitral, mitral annular and tricuspid regurgitation velocities. The only study that compared diastolic KE was from Al-Wakeel et al.  and showAs a relatively new technology, 4D flow CMR is not yet widely accessible, and expertise are not widespread . FDA appAcquisition time can be reduced by eliminating respiratory motion compensation, but this can significantly impact image quality . Self-gaIn 4D flow CMR, the normal practice of having a single velocity encoding range (or VENC) can result in an insufficient velocity to noise ratio (VNR) in regions with low velocities. Techniques that enable multi-VENC measurements allow for a better VNR over the whole velocity range, but at the cost of a longer acquisition time .Prior to 4D flow data analysis, pre-processing to correct for phase offset errors such as eddy current effects and velocity aliasing is necessary. For this, several commercial FDA approved and CE marked software solution are available, but adequate technical expertise is still required as optimal strategies may vary according to MR system, 4D flow CMR sequence and application . AdequatData analysis and 4D flow visualization techniques, like acquisition and processing methods, may differ from center to center, and not all parameters can be measured in all centers. As a result, a variety of different parameters have been used across studies, meaning that results are often not directly comparable, and some studies have had conflicting results , 73.Lastly, 4D flow CMR generates huge data sets and therefore adequate data management, and storage infrastructure are essential to be able to process 4D flow data.Further, more comprehensive, validation of 4D parameters against existing non-invasive and invasive markers is required, and ideally linked to clinical outcomes to determine the true utility of these techniques. Testing at different field strengths is also required, as is cross-vendor standardization of acquisition, processing, and analysis techniques, before 4D flow can be more widely used clinically for assessment of diastolic function—as outlined in the 2015 Society for Cardiovascular Magnetic Resonance (SCMR) consensus statement .Research into the assessment of LV diastolic function using 4D flow CMR has so far consisted almost entirely of small single center studies. Larger multicenter and multimodality studies are necessary for comprehensive assessment of HFpEF (and other cardiac conditions involving LVDD), and more definitive comparisons of the performance of novel 4D parameters against contemporary diagnostic and prognostic markers of disease.Early studies of LVDD using time resolved 3-dimensional velocity mapping acquired with 4D flow CMR have contributed to our understanding of complex physiological and pathophysiological processes such as energy loss, vortex formation and functional blood flow components . Novel 4ZA, SM, C-JC, TE, and BR made substantial contributions to the concept and design of the manuscript. All authors including SN made substantial contributions to the drafting and revision of the manuscript. All authors have 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.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.


Pragmatically identifying persons living with dementia (PLWD) for research is a persistent challenge, reflecting limitations in data elements. As part of the D-CARE study, the recruitment coordinator and analyst collaborated to create a screening algorithm to identify potentially eligible patients. A combination SAS/R script was applied on a monthly basis (Oct 2020-Dec 2021) to a clinical data warehouse, identifying living patients 50 years or older with ICD-10 codes for dementia or related conditions within the past two years, and an encounter within the past year. This process identified 6,478 unique patients, leading to the recruitment of 837 PLWD into the study (22% Black or Hispanic). Algorithmic components that enhanced recruitment efforts included the incorporation of data elements necessary to contact and enroll patients, formatting data consistent with reporting requirements, and tracking information on primary care provider, diagnosis dates with the set of providers that agreed to recruit for the trial.


Adequate sleep especially during developmental stages of life, is considered essential for normal brain development and believed to play an important role in promoting healthy cognitive and psychosocial development, while persistent sleep disturbances and/or sleep deprivation during early life are believed to trigger many mental ailments such as anxiety disorders, depression, and cognitive impairment. Initially it was suggested that adverse mental health conditions adversely affect sleep, however, it is now accepted that this association is bidirectional. In fact, sleep disturbances are listed as a symptom of many mental health disorders. Of special interest is the association between early life sleep deprivation and its negative mental health outcomes. Studies have linked persistent early life sleep deprivation with later life behavioral and cognitive disturbances. Neurobiological underpinnings responsible for the negative outcomes of early life sleep deprivation are not understood. This is a significant barrier for early therapeutic and/or behavioral intervention, which can be feasible only if biological underpinnings are well-understood. Animal studies have provided useful insights in this area. This article focusses on the knowledge gained from the research conducted in the area of early life sleep deprivation, brain development, and behavioral function studies. A question often arises during discussions of sleep, “How much sleep is good sleep?” The National Sleep Foundation (NSF) and the American Academy of Sleep Medicine (AASP)/Sleep Research Society (SRS) both have offered their recommendations. For example, infants between the ages of 4–11 months are recommended 12–15 h of sleep by NSF while AASP/SRS recommends 12–16 h of sleep. Children between the age of 6–13 years are recommended 9–11 h by NSF and children from ages 6–12 years are recommended 9–12 h. Teenagers are recommended 8–10 h by both organizations. NSF considers teenagers to be 14–17 year old while AASP/SRS classifies them as between 13 and 17 year old  data. PSG involves simultaneous recording of the activity from the cortical area of the brain using electroencephalogram (EEG), eye movement via electrooculogram (EOG), and electromyogram (EMG)-mediated recording of the skeletal muscle activity. In humans and other mammals, sleep consists of two main phases: rapid eye movement (REM) sleep and non-REM (NREM) sleep. NREM sleep, also known as slow wave sleep, is characterized by high amplitude low frequency EEG waves, decreased muscle tone, and slow eye movement. While REM sleep, known as active sleep, is characterized by low amplitude high-frequency waves, muscle atonia, and rapid eye movement. EEG slow wave activity (delta power) in NREM sleep is considered as a marker of sleep propensity. In humans, NREM sleep is further classified into N1, N2, and N3 stages. Each NREM stage has a well-defined EEG wave pattern. During sleep, the different phases of sleep alternate in a tightly regulated pattern resulting in creation of sleep cycles , coordinated by several defined nuclei in the pons and midbrain areas. Cells promoting wakefulness are; noradrenergic (NE) cells of the locus coeruleus, serotonergic (5-HT) cells of the raphe nuclei, cholinergic (Ach) cells of the pedunculopontine tegmentum and laterodorsal tegmentum, glutamatergic cells (Glu) of the midbrain, and dopaminergic (DA) cells of the substantia nigra and ventral tegmental area. Projections of wake-promoting cells activate thalamocortical, hypothalamo-cortical, and cortico-basal systems. Furthermore, there are 5 other groups of wake-promoting cells including; (1) the histaminergic cells of the posterior hypothalamic area, (2) the hypocretin/orexin cells of the lateral hypothalamus, (3) the cholinergic cells of the basal forebrain, (4) the neuropeptide Y (NPY) containing cells within the suprachiasmatic nucleus, and (5) the glutamatergic neurons present in the ventro medial prefrontal cortex (vmPFC). Activation of these systems maintains stable wakefulness by cortical activation and inhibition of activity in sleep-promoting areas of the brain. However, cell lesions within a particular wake-promoting group of cells does not by itself induce changes in the amount of wakefulness. This raises the interesting possibility that different brain regions collectively contribute to the promotion and maintenance of wakefulness, but none by itself are required for generation of wakefulness. Sleep physiology especially the understanding of the initiation and maintenance of sleep has been a challenge for decades and more research is needed. Initiation of sleep is suggested to be a result of successive passive and active physiological processes which leads to blockage of cortical sensory gates Datta, . The pasConsequently, cerebral cortex sensory signals at the cortical gate are blocked within the thalamus, depriving the cerebral cortex of receiving external information, as evident from high voltage and slow brain waves during NREM sleep, which is an indication of cortical inactivity  and related signaling molecules significantly in both the dentate gyrus and CA1 areas , GEAR, and SGP grants awarded to SS.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.