Among responders to microbial invasion, neutrophils represent one of the earliest and perhaps most important factors that contribute to initial host defense. Effective neutrophil immunity requires their rapid mobilization to the site of infection, which requires efficient extravasation, activation, chemotaxis, phagocytosis, and eventual killing of potential microbial pathogens. Following pathogen elimination, neutrophils must be eliminated to prevent additional host injury and subsequent exacerbation of the inflammatory response. Galectins, expressed in nearly every tissue and regulated by unique sensitivity to oxidative and proteolytic inactivation, appear to influence nearly every aspect of neutrophil function. In this review, we will examine the impact of galectins on neutrophils, with a particular focus on the unique biochemical traits that allow galectin family members to spatially and temporally regulate neutrophil function. A hallmark of effective immunity is the ability to rapidly recognize and respond to invading pathogens while avoiding potential injury to surrounding host tissue. This is especially important during the initial recruitment of neutrophils, one of the earliest and most effective responders to microbial infection . NeutropThe importance of appropriately governing early immune effectors is especially apparent in disease states in which neutrophil regulation is compromised. Genetic disease that impair neutrophil recruitment to sites of infection or reduce neutrophil effector activity leave patients prone to infectious disease –14. SimiWhile a variety of factors regulate neutrophil recruitment, activation and eventual removal, many studies have demonstrated that a series of carbohydrate binding proteins (CBPs) called galectins play a key role in this process. Galectin family members recognize highly modifiable cell surface carbohydrates to facilitate neutrophil extravasation, activation, microbial killing, and eventual turnover. While there have been many excellent reviews detailing the regulatory roles of galectins in general on immune activity and function –19, in t2+ to recognize cognate ligand, coined C-type lectins , neutrophils exhibited elevated expression of the S-type lectin Mac-2/Epsilon-bp  , a protetrophils . Gal-3 mtrophils .The ability of Gal-3 to regulate neutrophil activity through IgE engagement suggested that Gal-3 itself may influence neutrophil function. Subsequent studies demonstrated that Gal-3 can initiate neutrophil oxidative responses. In this setting, recombinant Gal-3 not only binds to neutrophils and stimulates superoxide production, but also directly activates neutrophils completely independent of IgE, in a carbohydrate- and dose-dependent manner . Gal-3 oEscherichia coli, Klebsiella pneumoniae, and Salmonella minnesota, possess this priming activity  neutrophils, implicating a role for priming events in the sensitization of neutrophils to Gal-3 . Primingactivity , suggestactivity . As Newcactivity , increasexposure .In addition to the impact of exposure to distinct microbial products or even microbes themselves on Gal-3 regulation of neutrophil activity, different disease and developmental states may also influence neutrophil sensitivity to Gal-3-induced ROS production. For example, while neonatal immunity is thought to be developmentally immature and less responsive to activating stimuli , neutropIn addition to inducing ROS, subsequent studies demonstrated that Gal-3 facilitates neutrophil activation, as evidenced by enhanced L-selectin shedding, increased CD11b expression and IL-8 secretion , 60. GalIn contrast to facilitating additional activation in the setting of infection, primed neutrophils can cleave Gal-3 into products no longer capable of signaling neutrophil ROS production, suggesting a potential negative feedback loop on Gal-3-mediated neutrophil activation. Importantly, cleaved, but not intact, Gal-3, is also preferentially internalized, raising the possibility that this cleavage event may also enhance Gal-3 removal . While Gin vivo in this process , though studies strongly indicate CD66a and CD66b are at least in part responsible for ROS induction by Gal-3 , 134. IgWhile many studies have defined galectin counter receptors on the surface of other immune cells, such as CD43 and CD45 on T cells, as the repertoire of glycosyltransferases can fundamentally differ between cell populations, these glycoproteins may or may not be decorated with suitable galectin ligands when expressed on neutrophils . As a reIt should be noted that while a given glycoprotein or glycolipid may serve as the functional receptor for a galectin or several galectin family members, it is certainly possible that galectins signal neutrophils through clustering of several similarly glycosylated receptors to ultimately induce a particular signaling outcome. Consistent with this possibility, Gal-3 clusters neutrophil counter receptors alone and in the context of adhesion to endothelial cells . LPS enhGiven the ability of galectins to broadly influence neutrophils, and in sometimes opposing manners, one critical challenge to the field is in understanding the dynamics of the regulatory network controlling galectin-glycan interactions to allow for proper control of neutrophil function. Clearly one key element is the expression of glycans on cognate receptors within the extracellular space. However, one often overlooked mechanism may be in the rich and fluctuating redox environment often accompanying inflammation, inflammatory resolution and eventual tissue repair.Protein oxidation is one of the strongest regulatory modifications linked to galectins, and is also one of the first identified. In fact, the identification of galectins remained elusive until it was discovered that their purification required reducing conditions , 26. In in vivo , leading to reduced cell surface expression in human breast cancer cell lines , it is also well-known that several proteolytic enzymes  are elevated during this time which could also serve to regulate galectin activity. Early studies showed that both MMP-2 (gelatinase-A) and MMP-9 (gelatinase-B) can cleave Gal-3 at specific residues within its N-terminal tail  and inhibit Gal-3 driven wound healing activity in T84 cells ; a variety of other MMPs have been implicated in altering Gal-3 expression/activity. MMP-7 , which is expressed in inflamed tissues often at the leading edge of gastrointestinal ulcers, was shown to cleave Gal-3 at three separate sites  was found to cleave Gal-3 in seminal plasma at Tyrt lectin , akin tot lectin . Regulatrd Gal-3 , 168. Rerd Gal-3 , this moThe distinct localization of galectins, their ability to selectively bind cell surface carbohydrates and their sensitivity to oxidative inactivation and proteolytic cleavage likely provided a unique evolutionary substrate to regulate the temporal and spatial activity of neutrophils during inflammation. Galectin expression within vascular endothelial cells and possibly in neutrophils themselves may contribute to extravasation, early activation and even chemotaxis . HoweverThe broad tissue distribution of galectins, coupled with their unique sensitivity to oxidative inactivation and proteolytic cleavage, may provide some insight into the temporal and spatial regulation of neutrophil function. Unlike most cytokines and chemokines, the vast majority of galectins reside in the cytosol, consistent with their lack of a signal peptide and consequential translation on free ribosomes . FollowiAs neutrophils effectively remove pathogens and necrotic tissue in the settings of inflammation, these cells can also infringe on surrounding viable tissue , 3. In cWhile there are a variety of distinct forms of programmed cell death, ranging from apoptosis to necroptosis , the abiThe ability of galectins to regulate neutrophil function suggests that these proteins may serve as useful pharmacological agents to favorably alter disease states marked by inadequate or exuberant neutrophil function. Consistent with this possibility, the earliest description of galectin-mediated immune regulation occurred following the exogenous delivery of electrolectin in a model of myasthenia gravis. Intriguingly, while the initial hypothesis was that delivery of electrolectin would stabilize the neuromuscular junction, additional experiments demonstrated that electrolectin actually inhibited the immune response required to induce myasthenia gravis –184. TheSubsequent studies demonstrated improved outcomes could also be achieved following galectin injection in additional models of immune-related pathology, including Concanavalin-A-induced hepatitis, collagen-induced arthritis, experimental autoimmune uveitis and experimental autoimmune encephalitis –187. TheThe vast majority of studies that have examined the impact of exogenous delivery of galectin on immune function have employed an intraperitoneal (IP) delivery route, a common approach of introducing substances in small rodent models, but one that is seldomly employed clinically , 189. ThInjection of galectins intravenously (IV) would appear to be a more favorable approach to avoid engagement of intraperitoneal contents and possibly model clinical routes of delivery more accurately. However, unpublished work by numerous labs has demonstrated that IV injection of active galectin-1 results in rapid death, presumably due to immediate galectin-induced hemagglutination and vascular stasis. While galectins have been reported to circulate, galectins detected as serum biomarkers of heart disease and other conditions likely represent inactive galectin as the assays employed in these studies utilize methods of antigen detection that do not directly assess galectin activity . As prevGiven the potential challenges of using galectins as modulators of neutrophil function clinically, alternative approaches may be required to fully harness the therapeutic potential of galectins to modify neutrophil function. Several reports have described various synthetic analogs of galectin ligands that appear to specifically inhibit distinct galectin family members , 193, prin vivo. As different galectins appear to regulate neutrophil function through similar pathways (in vivo studies have often been driven by initial in vitro observations and early in vitro data suggest that multiple galectins possess the ability to modulate neutrophil behavior in a similar manner, significant functional redundancy between different galectin family members in vivo may reduce the likelihood that a clear phenotype will be observed when using recipients deleted of only one or even several galectin family members. Furthermore, as galectins also regulate other immune populations (in vivo.Regardless of whether identifying and targeting galectin receptors will provide a suitable substitute for recombinant galectins as a therapeutic strategy, defining functional counter receptors for galectins will allow additional approaches to be used when seeking to further define the roles of galectins on neutrophil activity pathways –19, geneulations –19, whenin vivo studies appear to corroborate galectin activity on neutrophil function, additional studies are needed to formally test many of these hypotheses in vivo.Studies over several decades demonstrate that galectins can regulate a wide variety of neutrophil functions. The ability of galectins to bind a broad range of receptors and similarly be regulated by unique oxidative and proteolytic processes, suggests that evolution selected these unique immune regulators to temporally and spatially shape neutrophil function. In doing so, galectins appear to serve as critical regulators of neutrophil biology. While many 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.


Cardiovascular disease encompasses a wide range of conditions, resulting in the highest number of deaths worldwide. The underlying pathologies surrounding cardiovascular disease include a vast and complicated network of both cellular and molecular mechanisms. Unique phenotypic alterations in specific cell types, visualized as varying RNA expression-levels (both coding and non-coding), have been identified as crucial factors in the pathology underlying conditions such as heart failure and atherosclerosis. Recent advances in single-cell RNA sequencing (scRNA-seq) have elucidated a new realm of cell subpopulations and transcriptional variations that are associated with normal and pathological physiology in a wide variety of diseases. This breakthrough in the phenotypical understanding of our cells has brought novel insight into cardiovascular basic science. scRNA-seq allows for separation of widely distinct cell subpopulations which were, until recently, simply averaged together with bulk-tissue RNA-seq. scRNA-seq has been used to identify novel cell types in the heart and vasculature that could be implicated in a variety of disease pathologies. Furthermore, scRNA-seq has been able to identify significant heterogeneity of phenotypes within individual cell subtype populations. The ability to characterize single cells based on transcriptional phenotypes allows researchers the ability to map development of cells and identify changes in specific subpopulations due to diseases at a very high throughput. This review looks at recent scRNA-seq studies of various aspects of the cardiovascular system and discusses their potential value to our understanding of the cardiovascular system and pathology. Many possibilities for future applications (of precision medicine) spring to mind: today's blood counts might be replaced by a census of hundreds of distinct types of immune cells …”“—F. Collins and H. Varmus (2015)There has been tremendous curiosity to further understand the effect of phenotypic variation in the cardiovascular system and its relation to disease progression. For example, the increased expression of certain long non-coding RNAs and the reprogramming of fetal-heart genes are found to be crucial pathologic processes in the development of heart failure –4. The tWith the development of advanced RNA-seq techniques to better understand the transcriptome of tissues and individual cells, various groups have been able to produce novel insight in the native heart and associated pathology –5. RNA-sscRNA-seq has given us the ability to profile the phenotypes of single cells leading to discoveries of new cellular subpopulations that could contribute to cardiovascular pathogenesis. However, because of the complexity of the data, scRNA-seq findings may be difficult to interpret by clinicians or fit into current knowledge bases , 13. ThiCompared to the traditional technique of bulk-RNA-seq , the maiWhile there are numerous methods for performing scRNA-seq, all of which follow a general workflow: (1) isolating single cells, (2) capturing RNA, (3) reverse transcribing RNA to cDNA, (4) amplifying the cDNA, (5) library preparation, and (6) sequencing. It is within each of these steps that the variety of scRNA-seq protocols make use of different available technologies and methodologies, which are specifically advantageous to particular experimental designs and goals –15. For Despite varying advantages of different scRNA-seq protocols, there are shared challenges. For instance, accounting for library preparation costs and confounding batch effects remain significant barriers for all scRNA-seq protocols. One current improvement to scRNA-seq protocols which addresses these limitations is multiplexing . Recently developed techniques include: (1) lipid oligonucleotides which contain sample-level barcodes that incorporate into the plasma membrane of live cells, (2) combinatorial barcoding, where cells are identified through the unique combination of barcodes acquired from multiple rounds of random barcoding, and (3) using naturally variant single nucleotide polymorphism genotypes to distinguish cells' sample of origin, among others –22.Most centrally, these many scRNA-seq methods all function in accomplishing the same aim: to profile which genes are expressed and their level of expression for individual cells, which allows for novel data analyses important for investigating fundamental biological questions.Regardless of the exact protocol followed, all scRNA-seq data present unique interpretational challenges because of the high levels of technical and biological noise , 23, 24.The data analysis workflow for scRNA-seq data, as implemented by software packages like Cell Ranger Seurat, and Monocle, includes: (1) elucidating cells' heterogeneity via clustering cells based on gene expression profiles, (2) characterizing cell clusters by assigning cell type or functionality via biomarkers or differential expression analysis, and (3) organizing defined cell types/states into a trajectory  31–35).–35.31–35The processed scRNA-seq data is then suitable for use in analysis applications, such as, characterizing the cell clusters, and trajectory inference. The most straightforward method for characterizing cell clusters is to identify cluster specific expression of cell type biomarker genes. In the case where cells cannot be identified via biomarkers, differential expression analysis, of which the different methods are highly abundant and well-established, presents an alternative method for cell cluster characterization . DiffereWhile scRNA-seq data require careful processing to achieve interpretability, the end result of elucidating the cell types/states present in a sample and their relationships to each other  has critical implications for deepening our understanding of disease pathologies.Many studies prior to the utilization of scRNA-seq have demonstrated the importance of non-traditional cells such as resident fibroblasts, monocytes, and other non-cardiac myocytes within the cardiac network , 47. HowThis subsequent extracellular fibrosis formation following myocardial injury may be reparative or reactive . It has in vivo demonstrated a similar discovery of transcriptome variation in the human cardiac cellulome. The human embryo study identified spatially- and temporally-associated transcriptomic patterns of cardiomyocytes and fibroblasts during development  in rats (in situ hybridization (smFISH) with RNAscope to visualize the genetic expression variation within the myocardial tissue. smFISH with RNAscope brings significant clinical value by providing single molecule assessment of RNA biomarkers with less technical difficulty, higher sensitivity and higher specificity when compared to prior in situ RNA hybridization techniques  in a murine model  allows for the development of  disease . Studies disease . Howevernterpart , 75. Thenterpart . They idntiation . For exantiation .Single-cell resolution mapping has been applied to coronary vasculature development as well. It is known that the heart contains a vastly heterogenous microvascular network, and knowledge surrounding this network and its implication in disease remains limited . This heAs sudden death from myocardial infarction (MI) has decreased and more people are surviving MIs, there is a growing clinical need to attenuate the damage following MI to improve outcomes and prevent heart failure. Various strategies have been employed to reduce infarct size and preserve or even improve cardiac contractility –90. NeovAtherosclerosis has long been identified as an inflammatory response within the subendothelial space of arteries. However, atherosclerotic plaques are extremely heterogenous with different types of plaque formation, different growth rates, and differences in susceptibility to rupture . FurtherIn addition, phenotypic mapping by scRNA-seq was able to further elucidate the transcriptional overlap between macrophages and dendritic cellular phenotypes, which has been shown to contribute to early atherosclerotic formation , 110. ThAmong the various phenotypic expressions of different macrophage subtypes, it was shown in one scRNA-seq study that non-foamy macrophages expressed more of an inflammatory phenotype when compared to foamy macrophages . Non-foaSca1 expression could indicate an intermediate step for VSMC plasticity in plaque development. Although Sca1+ cells have been identified in bulk-blood vessel tissue before, scRNA-seq allowed for the identification of Sca1+ in a rare subpopulation of VSMCs, therefore providing higher-resolution insight into atherogensis  demonstrated that genetic variants of Tcf21 is causally associated with coronary artery disease, however, scRNA-seq may have elucidated a significant role for Tcf21 in phenotypic modulation (in vivo in the setting of coronary artery disease. The authors concluded that sinceTcf21 was associated with reduced risk of coronary artery disease, and that Tcf21 expression increased phenotypic modulation, phenotypic modulation is most likely associated with reduced coronary artery disease. It can be hypothesized that Tcf21 promotes VSMC to fibromyocyte transition thus augmenting fibrosis formation within the atherosclerotic lesion and the fibrous cap. This theoretically would stabilize the plaque from rupturing leading to an acute cardiovascular event. Although further studies need to be performed to investigateTcf21's atheroprotective potential, promoting Tcf21 activity could be an interesting approach to reduce the incidence of plaque rupture.Based on the findings from Dobnikar et al. upregulation of dulation . Phenotydulation . It has dulation . It has dulation , 122. Ho markers –125. Rec markers . This ph markers . VSMCs wdulation , 127. ThWe should note that many researchers have identified potential limitations to scRNA-seq data. It has been pointed out that the samples utilized for scRNA-seq do not capture the longitudinal expression of genes within the cardiac cellulome and that stochastic changes overtime could cause significant transcriptional variation , 129. ThThough, as was seen with the development of next generation sequencing, the current peak interest in single-cell technologies will result in rapid advancements in the field leading to more widespread use. Therefore, current cardiovascular scientists and cardiologists should become aware of its potential impact to the future of cardiovascular medicine . ThroughFC and JI contributed equally to the creation of this review. FC, JI, TB, DP, KG, DL, DR, and PL were all contributors to various parts of the review and helped in the writing of the review.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.


Neurodegenerative diseases are characterized by neurodegenerative changes or apoptosis of neurons involved in networks, leading to permanent paralysis and loss of sensation below the site of the injury. Cell replacement therapy has provided the basis for the development of potentially powerful new therapeutic strategies for a broad spectrum of human neurological diseases. In recent years, neurons and glial cells have successfully been generated from stem cells, and extensive efforts by investigators to develop stem cell-based brain transplantation therapies have been carried out. We review here notable previously published experimental and preclinical studies involving stem cell-based cell for neurodegenerative diseases and discuss the future prospects for stem cell therapy of neurological disorders in the clinical setting. Steady and solid progress in stem cell research in both basic and preclinical settings should support the hope for development of stem cell-based cell therapies for neurological diseases. Neurodegenerative diseases, such as Parkinson’s disease (PD), stroke, Huntington’s disease (HD) and amyotrophic lateral sclerosis (ALS), are characterized by neurodegenerative changes or apoptosis of neurons involved in networks, leading to permanent paralysis and loss of sensation below the site of the injury . UnfortuMeanwhile, human embryonic stem cells (ESCs) and adult stem cells have been coaxed into types of cells that repair neurodegenerative diseases insulation and replace nerve cells in neurodegenerative diseases . The potHere we review the scientific basis of stem cell therapies and discuss their prospects in Parkinson’s disease, Huntington's disease, Alzheimer's disease, Amyotrophic Lateral Sclerosis, stroke and spinal cord injury. In each of these neurodegenerative diseases, we describe the ways in which stem cells might be used to treat these conditions, discussing the prospects and problems of translating laboratory findings into clinically useful therapies.l-dopa and dopamine receptor agonists, and on deep-brain stimulation in the sub-thalamic nucleus [Parkinson’s disease is a neurodegenerative disorder characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and a reduction in striatal dopamine. In PD, the loss of DA neurons in the SNpc leads to impaired information processing in the basal ganglia ,7,8. The nucleus . Althoug nucleus ,12. ThusStudies show that stem cells overexpressing neurotrophic factors are able to induce neuroprotective and neuroregenerative effects after grafting in animal models ,14. ReceFor DA neurons generated from human ESCs and NSCs, survival after transplantation in animal models has been poor and needs to be markedly increased before clinical application will be possible . One potIn addition, the effects of growth factors on embryonic DA neuron grafts were recently studied ,24. The Huntington’s disease is a fatal, intractable disorder that is caused by polyglutamate expansions in the huntingtin protein ,27,28,29Most of the recent work in stem cell therapy has been conducted in animal models of HD. The study showed that cell replacement using grafts of fetal striatal neurons promoted functional recovery, and some evidence from clinical trials indicates that this could also occur in patients . The stuHowever, protocols and procedures developed from trials of fetal-derived cell transplantation in humans with HD lay the ground-work to move stem cell therapy into the clinic . One of in vitro [The grafts were also functionally beneficial given that the animals showed improvement in rotational behavior. In HD, mitochondrial defects are thought to play a significant part in the pathomechanism for cell death in HD pathology. Administration of 3NP, which inhibits mitochondrial succinate dehydrogenase (SDH), can induce the behavioral and anatomical features of HD in rodents and primates. Moreover, data demonstrated that stem cell factor (SCF), produced in situ in the lesioned striatum, was an important factor in promoting the engraftment of stem cells within the lesioned brain which could be able to activate the SCF receptor c-kit and its signaling pathway and to promote the migration and proliferation of mesenchymal and neural stem cells in vitro ,49,50.Taken together, neural implantation of stem cells may be of benefit in HD but a number of parameters of dose, treatment schedule, and route of administration need to be optimized.Alzheimer’s disease (AD) is characterized by neuronal and synaptic loss throughout the brain, involving the basal forebrain cholinergic system, amygdala, hippocampus and several cortical areas ,52,53,54in vitro and in vivo. A morphological abnormality of neurally-differentiated NSCs has also been described, which we too have seen in NSCs transfected with wild-type APP [Alzheimer’s disease amyloid precursor protein  has beentype APP . Althougtype APP , it is ttype APP ,67. Furttype APP .Because stem cells can be genetically modified to carry new genes and have high migratory capacity after brain transplantation, they could be used in place of fibroblasts that are known for their immobility following transplantation for delivery of nerve growth factor (NGF) to prevent degeneration of basal forebrain cholinergic neurons ,70. HoweAmyotrophic Lateral Sclerosis is an adult-onset neurodegenerative disorder characterized by degeneration and loss of motor neurons in the cerebral cortex, brainstem and spinal cord, leading to fatal paralysis ,75. A stin vitro from stem cells of various sources, including ESCs and those from the fetal CNS [in vitro, and extend axons to ventral roots after transplantation into adult rats, but whether these neurons can integrate into existing neural circuitries and restore motor function has not been established. Whereas neuronal replacement in ALS patients seems a distant goal, using stem cells to prevent motor neurons from dying is a more realistic and shorter-term clinical approach. This prospect is supported by studies showing that human embryonic germ cells delivered into the cerebrospinal fluid of rats with motor neuron injury can migrate into the spinal cord and induce motor recovery, probably through neuroprotection [Recent studies have indicated that it is possible to generate motor neurons in culture from stem cells that include ESCs and NSCs. Mouse ESC-derived motor neurons transplanted into motor neuron-injured rat spinal cord survived and extended axons into ventral root, and human ESCs transplanted into cerebrospinal fluid of rats with motor neuron injury migrated into spinal cord and led to improved motor function ,79,80. Retal CNS . Mouse Eotection ,83,84. TIt is unrealistic to expect that the transplantation of stem cells or stem cell-derived motor neurons in ALS patients in a clinical setting replaces lost neurons, integrates into existing neural circuitry, and restores motor function. Rather, preventing cell death in host motor neurons via provision of neurotrophic factors by transplanted stem cells or stem cell-derived motor neurons is a more realistic and achievable approach ,86,87. Fet al. showed that intrathecal injection with an optimized cell number could be a potential route for stem cell therapy in ALS patients. They suggested that at this dose of 1 × 106, the average number of motor neurons was significantly higher than others, and most injected hMSCs distributed in the ventricular systemand subarachnoid space [in vitro and in vivo studies have shown generation of motor neurons from human ESCs and functional engraftment of these motor neurons after transplantation into the developing chick and adult rodent spinal cord with axonal outgrowth toward muscle [The recent breakthroughs in stem cell research might nevertheless provide possibilities for neural implantation and cell replacement therapy for patients with ALS. Kim id space ,93. Addid muscle ,95,96,97d muscle . Once stroke damage has maximized, little can be done to recover premorbid function. In addition to therapies aimed at improving cerebral blood flow, there has been increasing emphasis on neuroprotective strategies. Recent attention has focused on restoring brain function through cell transplantation ,102,103.A recent study has reported that, in humans with ischemic infarct, intracerebral implantation of human teratocarcinoma NT2-derived neurons has resulted in functional improvement As a first step towards this goal, human fetal NSCs were transplanted into the brains of stroke-damaged rats, resulting in the migration of new neurons towards the ischaemic lesion . Other sThe therapeutic efficacy of such strategies could be improved further by genetically modifying the stem cells, for example, by overexpressing an anti-apoptotic gene. Interestingly, the stroke-damaged adult rodent brain has some capacity for neuronal replacement from its own NSCs . The disSpinal cord injury (SCI) invariably results in the loss of neurons and axonal degeneration at the lesion site, leading to permanent paralysis and loss of sensation below the site of the injury. It is interrupt ascending and descending axonal pathways, and cause a loss of neurons and glia, inflammation and demyelination . et al. showed that expression of caspase-3 on both neurons and oligodendrocytes after SCI was significantly down-regulated by MSCs [Preclinical studies have been performed on rats with a spinal cord injury and have shown that transplanted MSCs in the injured spinal cord survive, migrate into the host tissue and lead to axonal regeneration and motor function recovery . Dasari  by MSCs ,115. And by MSCs . ex vivo cell and gene therapy. Ronsyn et al. investigated the feasibility of a plasmid-based strategy for genetic modification of human MSCs with enhanced green fluorescent protein (EGFP) and neurotrophin (NT) [et al. found that MSCs delivered by lumbar puncture (LP) reached the contused spinal cord tissues and exerted a significant beneficial effect by reducing cyst and injury size [In addition, MSCs are attractive targets for hin (NT) . Bakshi ury size . Astrocyury size . With reury size . Althougury size . This review has discussed the major issues associated with stem cell therapy by transplantation for neurodegenerative diseases. Stem cells from a variety of sources have shown effectiveness in improving motor function after neurodegenerative diseases in animal experiments and clinical trails. Cell therapies in neurodegenerative disease are intended to protect neuronal populations susceptible to disease and replace dysfunctional or dying neurons. The use of both stem cell and growth factor–based therapies, although in its early stages, appears likely to contribute to future clinical strategies, including, but not limited to, neuroprotective and neuron replacement approaches. However, factors that control the differentiation, survival, and maturation of stem cells in the context of a host degenerative brain must be more thoroughly understood before stem cell therapy will prove to be a robust and safe strategy that can be transferred to the clinic. Furthermore, long-term and large scale multicenter clinical studies are required to determine further the precise therapeutic effect of stem cell transplantation.


Citrus aurantium or Citrus sinensis essential oils (EOs). The aim of this review is to analyze preclinical and clinical studies investigating the anxiety effects of Citrus aurantium or Citrus sinensis EOs on anxiety disorders. Clinical studies reporting the antianxiety effects of products containing Citrus aurantium or Citrus sinensis EOs in combination with other active substances, including medicinal plants, were excluded. Nine clinical studies fulfilled the criteria adopted for analysis. The bibliographic research was made on the major scientific databases. Analysis included only articles written in English and published on peer-reviewed scientific journals describing preclinical experiments and clinical trials carried out to investigate the antianxiety effects of Citrus aurantium or Citrus sinensis EOs produce anxiolytic effects both in preclinical experiments and in different clinical conditions. Citrus aurantium EO aromatherapy reduced anxiety level in the great part of stress conditions studied (subjects affected by chronic myeloid leukemia and preoperative patients) except for a sample of patients subjected to colonoscopy. Exposition to Citrus sinensis EO in clinical studies shows to be positive in reducing anxiety level in patients waiting for dental treatment as well as in healthy volunteers submitted to an anxiogenic situation. Data show that Citrus aurantium or Citrus sinensis on people with anxiety showed that inhalation or oral administration of Citrus aurantium and inhalation of Citrus sinensis can exert beneficial effects on anxiety; however, because of incomplete accuracy in the reporting of methodology, further more complete clinical studies are warranted. Overview of clinical trials conducted with Citrus are largely interbreedable. Among the common names given to the various members of the citrus family, orange often refers to the most popular Citrus sinensis and Citrus aurantium [ Citrus plants is characterized by the presence of several polyphenolic classes, including flavones, flavanones, flavonols, flavans, and anthocyanins [Citrus plants derived from the single genusurantium . Chemicaocyanins , 3.Experimental evidences highlight their pharmacological effects including antioxidant, cardioprotective, anti-proliferative, anticancer, and hypolipidemic activities –8.In folk medicine, products derived from the peel and/or whole dried immature fruit of orange plants have been used to treat several health problems such as gastrointestinal disturbances, respiratory disorders as agent for cough –12, inso Citrus bergamia have been described for their effects against stress, psoriasis, and hyperlipidemia [ Citrus aurantium and Citrus sinensis.Other citrus species such asipidemia . The preCitrus aurantium L., also called Seville orange, sour orange, or bitter orange, is a small citrus tree, about five meters tall, with scented white flowers, belonging to Rutaceae family, originating in eastern Africa, Arabia, and Syria, and cultivated in Spain, Italy, and North America [ America , 15.Citrus aurantium is called with several local common names in different countries where it is used for food, fragrance, and medical application. Fruit, peel, leaves, flowers, seeds, and essential oil (EO) of Citrus aurantium are used in perfumes and cosmetics, as well as in the food and confectionery industry [ Citrus aurantium octopamine, hordenine, tyramine, and N‐methyltyramine are absent or in trace amounts in bitter orange extracts [industry . Bitter industry . The comindustry –20. Flavindustry . Other mextracts –25. Citrus aurantium are widely consumed in dietary supplements for appetite control, weight management, sports performance, and energy, and bitter orange products are also consumed in the form of food as juices and marmalades [Standardized aqueous-alcoholic extracts of the immature fruits ofrmalades , 26.Citrus aurantium EO, also known as neroli oil, is widely used in aromatherapy. It has been suggested that it stimulates central nervous system, lowers blood pressure, and has sedative, analgesic, anti-inflammatory, antispasmodic, carminative, digestive, and diuretic effects [ Citrus aurantium fresh lives [ effects . It is ash lives .Citrus sinensis L., named orange or sweet orange, is a millennial small tree belonging to the Rutaceae (citrus) family originated in southern China. The orange tree is small, spiny tree, typically growing to 7.5 m, but occasionally reaching heights up to 15 m, generally with a compact crown. Orange tree grows in tropical, semitropical, and warm temperate regions, becoming the most widely cultivated fruit tree in the world [he world , 30. OraThe sweet orange tree is found more or less in the same places as the bitter orange tree. Sweet orange oil is extracted from the fruit of the tree via cold pressure; it is also possible to distill sweet orange oil .Citrus sinensis contains several active secondary metabolites contributing to the pharmacological activities of the plant. In Citrus sinensis fruits, peel, leaves, juice, and roots, several types of chemical compounds including flavonoids [avonoids , 32, hydavonoids , 33.C. sinensis is a rich source of vitamin C, a natural antioxidant that support the immune system activity [ C. sinensis has been used traditionally, to treat intestinal disorders , respiratory disorders , obesity, menstrual disorder, cardiovascular disease , anxiety, depression, and stress [activity , 34. C. d stress .Anxiety disorders are among the leading prevalent causes of global mental disorders , 37. The Citrus EO is able to influence the activity of brain areas such as the hypothalamus, hippocampus, and pyriform; preclinical and clinical research showed that citrus fragrance can restore stress-induced cortex [It is known that inhalation of volatile components ofd cortex , 41 and d cortex  and may d cortex , 44. Citrus aurantium and Citrus sinensis are really effective in the treatment of anxiety conditions. With this aim, we summarized the published reports of preclinical and clinical studies regarding the use of Citrus aurantium- or Citrus sinensis-based products in conditions related to anxiety disorders.In light of the abovementioned findings, we tried to assess if the potential health effects of bothCitrus aurantium axiety”, “Citrus sinensis anxiety”, and “clinical trials” were used. Analysis included only articles written in English and published on peer-reviewed scientific journals describing clinical trials and applications of Citrus aurantium or Citrus sinensis EOs. Articles describing the effects of products containing Citrus aurantium or Citrus sinensis EOs in combination with other active substances including medicinal plants derivatives were also excluded. The selection and the review of clinical studies written in English regarding Citrus aurantium or Citrus sinensis EOs were performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [A bibliographic research was carried out independently by two researchers  in the major scientific databases and search engines of peer-reviewed literature from 2000 to July 2018, on life sciences and biomedical topics . The following keywords or combination of keywords “Citrus anxiety”, “tatement . Methodotatement  and the tatement . From th Citrus aurantium or Citrus sinensis treatment for anxiety were corresponding to the inclusion criteria. 63 articles were excluded for title and abstract and 4 were excluded because the products object of the studies were combinations including Citrus aurantium.A collection of 94 scientific articles was selected from our bibliography research. Only 17 articles describing effects of Citrus aurantium or Citrus sinensis were administered for inhalation as aromatherapy, and in one study Citrus aurantium was orally administered.Nine clinical studies were included in the review . In eigh Citrus aurantium or Citrus sinensis administration. Tables  Citrus aurantium or Citrus sinensis, respectively. Tables  Citrus aurantium and Citrus sinensis, respectively.  Citrus aurantium were demonstrated through behavioral experiments carried out with laboratory animals. C. aurantium EO oral administration in mice increased exploration of the open arms (time spent) in the elevated plus-maze at a dose that did not impair motor activity observed with open-field and rotarod test, which is indicative of anxiolytic-like effect in male mice [Antianxiety effects ofale mice . Citrus aurantium EO administration [The acute administration induced an anxiolytic-like effect in the light/dark transition tests  and in marble burying (decreased number of marbles buried) without any motor impairment, while repeated administration showed effects in the marble-burying test only. Repeated diazepam administrations did not increase light/dark transitions. Thus, the results suggest an anxiolytic-like effect following acute and repeatedstration . Citrus aurantium EO was reversed by 5-HT1A antagonist WAY100635 but not by flumazenil, a benzodiazepine antagonist, suggesting serotonergic mediation [Similar results were obtained in another experiment showing that anxiolytic-like effect of oral administration ofediation . Citrus aurantium EO increased social interactions  in rats and increased exploration time in the open arms of the elevated plus-maze, suggesting an anxiolytic-like effect at a dose that did not impair motor activity in the open-field test [In another study, inhalation ofeld test . Citrus aurantium EO, similar to fluoxetine, increased open arm explorations (percentage of time spent and percentage of entries) in the elevated plus-maze in male mice. The authors suggested that the effect of Citrus aurantium L. is linked to serotonergic transmission based on a fluoxetine + Citrus aurantium EO interaction. However, Citrus aurantium EO did not change the anxiolytic effect of fluoxetine in the elevated plus-maze, suggesting no drug interaction [Acute intraperitoneal administration oferaction . Citrus aurantium EO on anxiety and its interaction with GABAergic pathways, evaluating the coadministration effects of Citrus aurantium and diazepam. Results showed that Citrus aurantium EO increased the open arms exploration (increase of percentage time spent) of male mice submitted to the elevated plus-maze. Although diazepam increased open arms exploration (percentage of time spent and percentage of entries), coadministration with Citrus aurantium reduced the anxiolytic effect of diazepam. Flumazenil did not alter the effect of Citrus aurantium; however, authors suggest that Citrus aurantium L. may exert an anxiolytic-like effect acting as partial agonist at the GABA-A receptor/benzodiazepine site [ Citrus aurantium are summarized in Khosrovi et al. investigated the effect of intraperitoneal injection ofine site . Preclin Citrus aurantium EO effects on anxiety levels during different medical conditions were found. All of the six studies in which Citrus aurantium was investigated were randomized/controlled clinical trials.Complex six studies investigating Citrus aurantium EO in reducing anxiety during labor in a group of Iranian pregnant women [One randomized clinical trial described the effects ofnt women . Citrus aurantium compared with the control group, thus suggesting that aromatherapy with Citrus aurantium EO could reduce anxiety during labor [Before the aromatherapy, both groups had the same levels of anxiety; the levels of anxiety evaluated at dilations of 3-4 and 6-8 cm were significantly lower in the aromatherapy group withng labor .A trial was carried out on patients proposed for colonoscopy and divided into two groups. Aromatherapy was performed by inhalation of Sunflower oil (control group) and Neroli oil . Results showed that there was no significance difference of procedural anxiety measured by State Trait Anxiety Inventory state (STAI-S) score and procedural pain evaluated by visual analogue scale (VAS), before and after aromatherapy in patients subjected to colonoscopy. Significant lower pre- and postprocedural systolic blood pressure in Neroli group than control group were observed. The authors concluded that the inhalation aromatic agent had an effect on lowering procedural anxiety-related  blood pressure .Patients with a mandibular third molar with B II classification of impacted teeth and American Society of Anaesthesiologists (ASA) class I patients , with moderate and high anxiety levels measured through the dental anxiety scale (DAS) questionnaire, were included in another randomized and controlled study.The ASA clinical status classification system assessed the fitness of patients before surgery. The outcome variables were physiologic measures related to anxiety, including mean blood pressure, respiratory rate, and pulse rate.After aromatherapy, mean blood pressure, pulse rate, and respiratory rate were significantly lower in the fragrance group during surgery . Vital signs measurements showed significant differences between the two groups, which may be due to the sedative effect of the orange fragrance during surgery. Results suggest that ambient orange fragrance could be helpful in reducing dental anxiety during dental surgical removal . Citrus aurantium EO with diazepam anxiolytic effects, in the moment that precedes the collection of medullary material in patients with CML. Systolic and diastolic blood pressure and cardiac and respiratory frequencies were measured. C. aurantium EO decreased both systolic and diastolic blood pressure while with diazepam, only systolic blood pressure decreased. The Citrus aurantium EO did not decrease the respiratory frequencies.Another study was carried out on a Brazilian volunteers' cohort with chronic myeloid leukemia (CML), treated with standard therapy. The study compared Citrus aurantium EO group of patients with CML but not in the CML diazepam and placebo groups. In conclusion, study results showed an anxiolytic effect of Citrus aurantium EO in patients with CML with an improvement of psychological and physiologic parameters. For the authors, this effect has a great clinical relevance, because patients with cancer go through various stressful phases during the disease, and the standard therapy significantly contributes to improve the anxiety level and physiological parameters during a procedure that is a cause of distress [The use of STAI-S revealed an anxiolytic effect ofdistress . Citrus aurantium EO in patients experiencing crack withdrawal. Based on the fact that individuals who experience crack withdrawal present a high anxiety trait, anxiety status was induced with the Simulated Public Speaking (SPS) method (subject is requested to deliver a speech in front of a video camera with its image being displayed on a TV screen). Anxiety levels were assessed by the Inventário de Ansiedade Traço-Estado (IDATE), the Brazilian version of STAI-S.Chaves Neto et al. (2017) studied the anxiolytic effects of Citrus aurantium EO maintained controlled anxiety levels during SPS when compared to the control group (no treatment). Subjects who were administered Citrus aurantium EO also maintained states of discomfort and cognitive impairment often associated with anxiety during SPS. However, nebulization of the EO of Citrus aurantium provided an acute anxiolytic effect in crack cocaine users exposed to SPS.The results demonstrated that subjects in the groups treated with Citrus aurantium EO, administered by nebulization, produced anxiolytic effects in crack users in abstinence thus indicating the possibility of Citrus aurantium EO use as an alternative complementary therapy in the control of anxiety in users who are abandoning drugs abuse [Authors' conclusions were thatgs abuse . Citrus aurantium blossom distillate (CABd) on preoperative anxiety was evaluated. Preoperative anxiety was assessed using STAIS and Amsterdam Preoperative Anxiety and Information Scale (APAIS). The main finding of this study was the confirmation of the anxiolytic effect obtained with oral administration of blossom distillate Citrus aurantium. Both STAI-state and APAIS scores were decreased by CABd. On the other hand, neither STAI-state nor APAIS was changed in the placebo group [In a randomized double-blind design, the effect of oral administration ofbo group . Citrus aurantium EO showed that inhalation of the oil produces significant anxiolytic effects. Methods for diffusion of EO used in the studies are by direct inhalation with hand-hold nebulizers generally with doses of EO ranging 10 – 50 mL or by dilution of EO in distilled water and successive diffusion through an electric dispenser or by gauzes impregnated with 4 mL of Citrus aurantium EO.Results of the studies using Citrus aurantium, a reduced number of preclinical and clinical experiments were conducted on Citrus sinensis. Inhalation of Citrus sinensis EO  in rats submitted to elevated plus-maze followed by the light/dark paradigm produced an increase in open arm exploration (% time spent and % number of entries) in the elevated plus-maze, and of the time spent in the lit chamber of the light/dark test. No effect on motor activity, as measured by the total distance travelled in the elevated plus-maze, was detected [In comparison to findings published about investigation ondetected . Citrus aurantium and Citrus sinensis EOs were evaluated through behavioral tests and measurement of corticosterone and melatonin plasma levels in mice.Anxiolytic‐like, sedative, and antidepressant‐like potential effects of inhalation of both Citrus sinensis EO 10% inhalation, without affecting melatonin and corticosterone physiological levels. Inhalation of 10% Citrus aurantium EO did not show neither anxiolytic-like effects nor change in melatonin and corticosterone plasma levels [Results of behavioural tests showed an anxiolytic‐like and sedative effect ofa levels . Citrus sinensis were found. Two studies describe the effects of Citrus sinensis EO on anxiety in adults and children, respectively, subjected to dental treatments.Three randomized and controlled studies investigating on the effects of Citrus sinensis. Orange odor was diffused in the waiting room of odor group through an electrical dispenser whereas in the control group no odor was diffused. For assessing cognitive functions, patients completed the Wortschatz test (WST) [A randomized controlled trial was carried out on adult patients waiting for dental treatment to evaluate the anxiolytic effects ofst (WST) . For assst (WST) . The Mehst (WST)  were use Citrus sinensis effects, showing that women exposed to sweet orange EO had a lower level of state anxiety, a more positive mood, and a higher level of calmness [Results of the study showed that exposure to ambient orange odor has a relaxant effect compared to control group. Results reported also a sex difference ofcalmness .Citrus sinensis) on child anxiety during dental treatment were also evaluated in another study by using salivary cortisol and pulse rate as index of patients' anxiety.The effects of orange odor , a method to induce anxiety. During the text, a board with 100 of the color-naming words blue, yellow, red, green, and violet organized randomly in a 10 · 10 matrix is presented to each participant. The color of each word is different from its own meaning . The participant has to say quickly (in two minutes) the sequence presented, the color of the ink, but not the colors described by the single words. The whole test is recorded and presented to the subject on a monitor during the test [The results of this study showed that the salivary cortisol level and pulse rate significantly decreased in intervention groups by using aromatherapy withensis EO . The potthe test .State-Trait Anxiety Inventory (STAI) and Visual Analogue Mood Scale (VAMS) were used to evaluate psychologic parameters. Physiologic parameters were evaluated before the inhalation period and before, during, and after the SCWT. Results showed dose-dependent anxiolytic properties of sweet orange EO . Citrus sinensis EO exerts anxiolytic effects in anxiogenic situations. One of the studies show that Citrus sinensis EO exerts its anxiolytic effects also in children.These three studies indicate that Citrus fragrances have mood-enhancing properties. These effects were confirmed by successful clinical study carried out with citrus fruits oils on patients affected by stress symptoms or depression [EOs physiological and psychological effects are known in folk medicine and aromatherapy for a long time , 66. Aropression , 68.Anxiety disorders are the most common type of psychiatric disorders in the general population . Their tCitrus aurantium and Citrus sinensis are rich in flavonoids and polyphenolic compounds with numerous pharmacological properties, such as the inhibition of the oxidation of low-molecular weight proteins and platelet accumulation thus contributing to immune cell stability.They are also used in treatment of mental disorders, inflammation, viral infections, and allergies , 74. Fla Citrus aurantium or Citrus sinensis for the treatment of anxiety. We analyzed all clinical studies according to the recommendations described in the checklist developed by the Consolidated Standards of Reporting Trials [ Citrus aurantium use was carried out with a double-blind methodology  except for a sample of patients subjected to colonoscopy. Exposition to Citrus sinensis EO in clinical studies is shown to be positive in reducing anxiety level in patients waiting for dental treatment as well as in healthy volunteers submitted to an anxiogenic situation. However, a definitive conclusion needs further studies because of the reduced number of studies and the small number of patients for each study. Regarding data reporting of clinical studies with Citrus aurantium, they appear without description of the characteristics of the herbal product and lack qualitative testing. From the methodological point of view, most of the clinical studies show the lack of important items such as treatment allocation conceal and description of double-blind procedure. On the basis of the present overview, we can conclude that the use of Citrus aurantium and Citrus sinensis EOs could be useful to reduce anxiety caused by clinical stress conditions; however, results are impaired by the poor accuracy and methodology applied in clinical studies.Antianxiety effects of


Schizophrenia (SCZ) is a common psychiatric disorder with polygenetic pathogenesis. Among the many identified candidate genes and loci, the group of tumour suppressor genes has drawn our interest. In this mini-review article, we describe evidence of a correlation between major tumour suppressor genes and SCZ development. Genetic mutations ranging from single nucleotide polymorphisms to large structural alterations have been found in tumour-related genes in patients with SCZ. Epigenetic mechanisms, including DNA methylation/acetylation and microRNA regulation of tumour suppressor genes, have also been implicated in SCZ. Beyond genetic correlations, we hope to establish causal relationships between tumour suppressor gene function and SCZ risk. Accumulating evidence shows that tumour suppressor genes may mediate cell survival and neural development, both of which contribute to SCZ aetiology. Moreover, converging intracellular signalling pathways indicate a role of tumour suppressor genes in SCZ pathogenesis. Tumour suppressor gene function may mediate a direct link between neural development and function and psychiatric disorders, including SCZ. A deeper understanding of how neural cell development is affected by tumour suppressors may lead to improved anti-psychotic drugs. Schizophrenia (SCZ) has a worldwide prevalence of approximately 1%  and MspI (intron 6) restriction sites were shown to confer greater susceptibility to lung cancer in Turkish patients with SCZ . Unique phenotypes are associated with different mutant alleles and loci. In genetic studies of SCZ, a number of different candidate loci have been identified. The relationship between p53 polymorphisms and SCZ was reported in an early study showing that patients carrying specific alleles had reduced vulnerability to lung cancer (Park et al., APC exons that are associated with SCZ, including rs2229992, rs42427, and rs465899 have been identified (Chambers and Perrone-Bizzozero, Additional candidate genes and loci associated with SCZ continue to be identified. A genomic study focusing on human chromosome 8p has identified dozens of risk loci, encoding both protein and microRNA in each region, including various tumour suppressor genes with structural variations, such as copy number variants, microdeletions, and microduplications, all of which contribute to SCZ (Tabarés-Seisdedos and Rubenstein, In addition to the direct regulation of protein expression and function by variants in protein-coding regions, epigenetic mechanisms play an important role in SCZ onset by regulating protein expression without altering the gene sequence. Evidence is continuously growing for the role of epigenetics in SCZ occurrence (Dempster et al., TP53 gene expression and leads to excessive neurite outgrowth (Ma et al., Although the abovementioned evidence highlights the correlation between tumour suppressor genes and SCZ susceptibility, we still lack evidence of a causal relationship. One possible hypothesis is that tumour suppressor genes participate in SCZ pathogenesis through neurodevelopment or neural functions. The first piece of supporting evidence for the above hypothesis was reported for the p53 gene, whose over-expression was shown to lead to excessive neuronal death and impaired neural function (Hughes et al., APC leads to development of age-dependent working memory deficits and hypoactivity (Koshimizu et al., APC gene has also been reported to result in abnormal dendritic spine formation and long-term potentiation in hippocampal neurons, and is associated with impaired social interaction (Onouchi et al., Other tumour suppressor genes may also contribute to SCZ pathogenesis due to their effects on neural development, including axon guidance and dendritic arborisation. They include SCZ risk genes such as disrupted in SCZ-1 (Miyoshi et al., AF1q, contributed to SCZ susceptibility (Su et al., SCZ pathogenesis is inherently a polygenic event involving interactions among multiple risk genes. A recent association study reported that the interaction between the SNP loci of two tumour suppressor genes, thioredoxin interacting protein and In addition to converging molecular pathways and transcriptional regulation between tumour suppressor and SCZ risk genes, shared epigenetic regulation of these two groups of genes may confer SCZ and cancer phenotypes. For example, age-related DNA methylation can elevate the risk of both SCZ and cancer (Jenkins et al., in vivo warrants more research and development as a potential pharmacological approach for SCZ. Histone deacetylase 2 is a critical mediator of atypical antipsychotic drug action (Kurita et al., We have summarised the recent major progress in understanding the roles of tumour suppressor genes in SCZ pathogenesis and discussed potential underlying cellular and molecular mechanisms. Tumour suppressor genes have been associated with neurodevelopmental disorders other than SCZ, such as autism Crespi, . These fBased on our current knowledge of tumour suppressor gene involvement in SCZ pathogenesis, we propose a working model in which at least certain SCZ cases can be attributed to functional hyperactivation of tumour suppressor genes, resulting in a concomitant reduction in susceptibility to cancers. Because tumour suppressor genes can inhibit cell proliferation and growth, they can also affect normal development or activity of neural tissues, thus leading to SCZ.CZhuo: study idea and design. HT, SL, and JL: systematic review, extraction, and analysis. FJ, CC, CL, MC, CZhou, LZ, and CZhuo: drafting of the manuscript. CZhuo, XS, JL, DW, and LZ: critical revision of the manuscript for important intellectual content. CZhuo, CC, CL, JL, LZ, and DW: access to data and had full access to all the data in the study and take the responsibility for the integrity of the data and the accuracy of the data analysis. All authors have reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer LC and the handling Editor declared their shared affiliation.


Eucalyptus, Populus, and various coniferous species. We discuss several appropriate experimental designs for extensive GWAS in a given undomesticated tree population, such as omics-wide association studies and high-throughput phenotyping technologies. We also explain why more attention should be paid to rare allelic and major structural variation. Finally, we explore the potential use of GWAS for the molecular breeding of trees. Such studies will help provide an integrated understanding of complex quantitative traits and should enable the molecular design of new cultivars.Wood formation is an excellent model system for quantitative trait analysis due to the strong associations between the transcriptional and metabolic traits that contribute to this complex process. Investigating the genetic architecture and regulatory mechanisms underlying wood formation will enhance our understanding of the quantitative genetics and genomics of complex phenotypic variation. Genome-wide association studies (GWASs) represent an ideal statistical strategy for dissecting the genetic basis of complex quantitative traits. However, elucidating the molecular mechanisms underlying many favorable loci that contribute to wood formation and optimizing GWAS design remain challenging in this omics era. In this review, we summarize the recent progress in GWAS-based functional genomics of wood property traits in major timber species such as  Wood, the secondary xylem of long-lived perennial plants, is produced via cell division from the vascular cambium, cell expansion, cell wall thickening, programmed cell death, and heartwood formation . In geneProgress has recently been made toward modifying the major wood biopolymers  in several model plants , but mucEucalyptus, Populus, and various coniferous species using forward genetic approaches, such as quantitative trait loci (QTLs) , lignin , cell wall sugars , and ultrastructural traits  in i (QTLs)  and candi (QTLs) . Howeveri (QTLs) . TherefoGiven the high genetic diversity, almost undomesticated status, rapid decay of linkage disequilibrium (LD), and minimal genetic structure of forest tree populations, such populations should represent ideal systems for conducting association studies and breeding using molecular marker-assisted selection (MAS). LD is fundamentally important for any genome-wide association study (GWAS) when genotyping does not cover all sequence variants in a genome. Indeed, the advantage of a low extent of LD is that once an association is detected, it is likely that this marker is physically close to the causal variant (likely within the gene itself) or is even the causal variant . DissectTrees have a wide geographical distribution and large wild population sizes and thus exhibit diverse responses to environmental changes. Association mapping principally exploits evolutionary recombination at the natural population level. Thus, a collection of cultivars/natural individuals with unknown ancestry and newly designed nested association mapping (NAM) populations are often used for association analysis in trees. The availability of fast, accurate estimation methods for variance components is a prerequisite for performing GWAS. Next-generation sequencing (NGS) and SNP arrays have opened up new possibilities for obtaining almost all SSRs and SNPs variants within a gene space or even within an genome-wide scan . MoreovePopulus trichocarpa) individuals and found 141 significant SNPs associated with cell wall traits. Only 40% of these associations involve genes previously known to function in wood formation  of functional relevance to cell wall recalcitrance in Populus  based on 180 wood-associated-genes, and the network hubs of several known NAC and MYB regulators, which were found using integrated GWAS and co-expression networks (Picea abies) . networks . The gena abies)  providesa abies) .P. trichocarpa accessions and found that hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase 2 (PtHCT2) controlled chlorogenic acid (CGA) and partially characterized metabolite levels, providing novel insights into omics-based inference of gene function in trees. Such practical applications of GWAS require the development of novel statistical methods, databases, and experimental designs for forest trees.Over the past 5 years, new whole-genome resequencing and emerging GWAS approaches that have been used to dissect ecologically relevant traits and wood traits in forest trees . Zhang eComplex phenotypic variations, such as wood formation in long-lived tree populations, involve a series of dynamic biological processes orchestrated in a precise, quantitative manner, including the transcriptional and translational regulation and the flux of metabolic intermediates of diverse biochemical pathways . We takeKnowledge of the genetic basis and hierarchical interactions of higher-order variation at an intermediate level, such as transcriptomic, proteomic, and metabolomic variation ranging from genotypic to phenotypic, has proven to be a great resources as “molecular phenotypes” will be crucial for dissecting the functional pathways underlying complex quantitative traits in trees Figure . The joiAs a prerequisite, precise sequencing and rigorous annotation of holistic reference genomes are needed for large heterozygous tree genomes, which remains challenging. Work is also needed to refine new sequencing technologies and to develop relevant bioinformatics approaches, which would lay the foundation for identifying genome-wide allele variation and performing massively parallel RNA sequencing (RNA-seq) of 1000s of genotypes . In partIn addition to obtaining complete genome sequence information, the improvement of phenotyping methods and the availability of high-quality trait data are also priority areas for successful GWAS in forest trees . Trees aMore attention should focus on how to develop high-throughput phenotyping (HTP) technologies to increase the precision of time-series data for functional plant traits. The emerging plant HTP platform uses a variety of imaging methods including visible imaging, imaging spectroscopy, thermal infrared imaging, fluorescence imaging, 3D imaging, and tomographic imaging to collect data for quantitative studies of complex traits related to plant growth, yield, adaptation, morphological and physiological traits .Such a computational ecosystem provides tremendous opportunities for solving foundational problems in predictive phenomics and accelerating breeding efforts . For exaOver the past few years, population-scale resequencing of the human genome has enabled more comprehensive analysis of allelic effects on certain complex diseases. Some rare variants  are likely to be more extreme than common variants for susceptibility to diseases with high and low heritability . Brachytic2 can moderately increase yield and reduce stem height in maize. The genomes of woody species have not experienced reductions in genetic diversity due to domestication, and rare variants are abundant among tree genomes but less tractable in GWAS. Such abundant rare variants are actually important for explaining the missing heritability of complex traits (Progress has been made in several crops and model plants. For example, x traits .Rare beneficial alleles are usually employed in current tree breeding, which once proven useful, undergo selective sweep and become fixed in all major cultivars. Balancing samples across population subdivisions and increasing the sample size can homogenize allele frequencies and elevate globally rare variants to common markers in some subpopulations. In addition, the use of multiple bi-parental crosses can break up population structure, which could increase the power of detecting multiple rare alleles that underlie natural variation. The promising approach of joint mapping with association panels and multiple bi-parental crosses in trees is also valuable for identifying low-frequency or small-effect alleles . DecreasAdvances in sequencing technology and bioinformatics algorithms offer the potential to test previously undetected structural variation within re-sequencing populations of a species . In contGenetic variation of quantitative traits is classified into additive, dominant, and epistatic effects, which are conferred by numerous genes/alleles in the multiple biological networks . It is lAdditionally, dissecting the genome-wide genetic basis of exhaustive gene/allele epistasis using different populations could provide insight into the roles of epistasis in response to local adaptation in trees . Once a A deeper knowledge of the genetic basis of complex traits, such as wood properties, can be achieved through the integrated use of diverse statistical models and experimental tools. Systems genetics could be used to understand the molecular mechanisms of candidate genes underlying traits of interest. Some promising studies demonstrate the advantages of this approach across a tree’s lifespan . HoweverTree MAS breeding may depend on the use of a pleiotropic allele or a favorable combination of alleles for multiple traits of interest , such asHigh-throughput GWAS combined with precision genome editing has huge potential for combining currently available causal alleles in the gene pool . This GADZhang planned the review. QD, DZhou, WL, MQ, and LX designed the figures and revised the review. QD, FS, PL, JX, and LW collected the data. QD wrote the paper. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A receptors and, by this mechanism, regulates emotional behaviors, is mainly synthesized in brain corticolimbic glutamatergic neurons. In PTSD patients, allopregnanolone down-regulation correlates with increased PTSD re-experiencing and comorbid depressive symptoms, CAPS-IV scores and Simms dysphoria cluster scores. In PTSD rodent models, including the socially isolated mouse, decrease in corticolimbic allopregnanolone biosynthesis is associated with enhanced contextual fear memory and impaired fear extinction. Allopregnanolone, its analogs or agents that stimulate its synthesis offer treatment approaches for facilitating fear extinction and, in general, for neuropsychopathologies characterized by a neurosteroid biosynthesis downregulation. The socially isolated mouse model reproduces several other deficits previously observed in PTSD patients, including altered GABAA receptor subunit subtypes and lack of benzodiazepines pharmacological efficacy. Transdiagnostic behavioral features, including expression of anxiety-like behavior, increased aggression, a behavioral component to reproduce behavioral traits of suicidal behavior in humans, as well as alcohol consumption are heightened in socially isolated rodents. Potentials for assessing novel biomarkers to predict, diagnose, and treat PTSD more efficiently are discussed in view of developing a precision medicine for improved PTSD pharmacological treatments.Post-traumatic stress disorder (PTSD) is a debilitating undertreated condition that affects 8%–13% of the general population and 20%–30% of military personnel. Currently, there are no specific medications that reduce PTSD symptoms or biomarkers that facilitate diagnosis, inform treatment selection or allow monitoring drug efficacy. PTSD animal models rely on stress-induced behavioral deficits that only partially reproduce PTSD neurobiology. PTSD heterogeneity, including comorbidity and symptoms overlap with other mental disorders, makes this attempt even more complicated. Allopregnanolone, a neurosteroid that positively, potently and allosterically modulates GABA Post-traumatic stress disorder (PTSD) is a multifaceted psychiatric disorder characterized by a high worldwide prevalence in the general population and a consistent global burden and disability. In the U.S., about 50%–85% of individuals during their lifetime experience traumatic events, of these, about 6.8% develop PTSD , is efficient in about half of the treated patients  that relate finding in PTSD patients to rodent stress or genetic models. Construct validity is the process involved in the onset and the manifestation of the disorder and this, ultimately, is recapitulated in the animal model. Finally, predictive validity reflects the capability of animals to inform by means of predictors on the human disorder.Establishing reliable biomarkers and specific treatments for PTSD has been hampered not only by the relative difficulty in establishing PTSD animal models but also because of the limited knowledge on PTSD neurobiology , forced swimming (20 min) and exposure to diethyl ether  are well characterized in anxiety, and a new model in mice having the fully VGV edited isoform of 5-HT2CR, which overexpresses brain 5-HT2CR, was recently established to study PTSD predisposition  or loneliness, elicits a number of physical, neurological and psychological deficits that range from Alzheimer’s disease to major depression, anxiety disorders and suicidality  during social isolation has also been one important research interest.Studies from this lab have mainly focused on the effects of social isolation on the GABAergic neurotransmission dysfunction caused by impaired neurosteroid biosynthesis, and changes in the expression of several GABAMice that are socially isolated for 3–4 weeks post-weaning (PN21) express a number of behavioral deficits relevant to model aspects of human mood disorders  and increase corticolimbic allopregnanolone levels  and only a few have quantified levels of allopregnanolone in the post-mortem brain  make even more challenging assessing a PTSD preclinical model. The socially isolated mouse model recapitulates several aspects of PTSD neurobiology, including downregulated corticolimbic allopregnanolone concentrations, changes in GABAThe author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hearing loss is one of the leading causes of disability in Canada and worldwide, with more than one million Canadians enduring a hearing-related disability. Meniere’s disease (MD) is a chronic condition of the inner ear, manifesting as a triad of disabling symptoms, including attacks of vertigo, fluctuating sensorineural hearing loss (SNHL), and tinnitus. Impacts on quality of life are severe, particularly with respect to restrictions in social participation and physical activity, fatigue, and reduced capacity to work. Anxiety and other psychological disorders may result from the restrictions imposed on life, the constant uncertainty of vertigo attacks, and fluctuating SNHL, with neuroses and depression affecting 40 to 60% of sufferers of intractable MD. There is a need to establish the benefits of previously studied interventions with greater certainty. The planned systematic review and meta-analyses/network meta-analyses (NMAs) will assess the relative effects of competing pharmacologic and surgical interventions for management of MD in adults.An experienced medical information specialist in consultation with the review team will develop the electronic search strategies. We will search various databases including MEDLINE, Embase, and the Cochrane Library with no date or language restrictions for published literature, and key clinical trial registries for in-progress and completed trials. Screening of the literature will be performed by two reviewers independently using pre-specified eligibility criteria, and quality of the included studies will be assessed using the Cochrane Risk of Bias tool. We will resolve disagreements through consensus or third-party adjudication. When applicable, meta-analyses and NMAs will be pursued to compare interventions in terms of their effects on outcomes, including frequency and severity of vertigo, occurrence and intensity of tinnitus, changes in hearing and speech recognition, quality of life, and harms. Separate analyses exploring the effects of pharmacologic and surgical approaches will be performed.Our planned systematic review will provide informative evaluations of existing treatments for management of Meniere’s disease. The findings will inform practitioners as to the relative benefits and harms of the existing competing interventions for MD, offer optimal clinical treatment strategies, identify evidence gaps, and determine promising therapies for evaluation in future trials.CRD42019119129PROSPERO  Hearing loss is one of the leading causes of disability in Canada and worldwide, with more than one million Canadians enduring a hearing-related disability , 2. DespMD is a chronic condition of the inner ear, manifesting as a triad of disabling symptoms, including attacks of vertigo, fluctuating sensorineural hearing loss (SNHL), and tinnitus . ImpactsThe detrimental effects of MD on patients’ quality of life may be severe, and thus early detection and rapid intervention are encouraged . HoweverThe primary objective of this systematic review is to assess the relative effects of available pharmacologic therapies in patients with MD on vertigo and other key patient outcomes in randomized controlled trials and quasi-randomized trials. The secondary objective is to assess the effects of surgical interventions in the MD population on vertigo and other key patient outcomes in randomized and quasi-randomized trials.This protocol adheres to the standards of the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) Statement , and is Population. Studies enrolling adult patients with MD per established criteria  ) receivi. The following interventions will be of interest:○ Systemic pharmaceuticals: diuretics , Motion sickness/anti-nausea medications , phenothiazines), benzodiazepines ;○ Intra-tympanic pharmaceuticals: intra-tympanic gentamicin, intra-tympanic steroids;○ Surgical interventions: sacculotomy, vestibular nerve section, labyrinthectomy, tympanostomy tube, endolymphatic duct blockage, endolymphatic sac decompression, endolymphatic shunt, transtympanic pressure treatment.Interventions/comparatorsWe have established the review eligibility criteria based on the PICOS (Population-Intervention-Comparators-Outcomes-Study design) framework. We will include primary studies that meet the following criteria:○ frequency, severity, type, and control of vertigo measured via electrocochleography score test or other methods;○ occurrence and intensity of tinnitus measured via various methods such as psychoacoustic tests , rating scales , questionnaires describing functional effects , and patients’ global perception of treatment-related changes;○ changes in hearing, based on Pure Tone Average (PTA) in decibels, and speech recognition, such as word recognition score (WRS) that may also be labeled as speech discrimination score (SDS), and speech reception threshold (SRT);○ quality of life measured by various scales such as Quality of Well-being Scale (QWB), SF-12, Physical SF-12 score; Mental SF-12 score, Center for Epidemiologic Studies–Depression Scale (CESD);○ perception of aural fullness;○ and harms, including hearing loss, withdrawals due to adverse effects, and serious side effects defined by authors.Outcomes. Endpoints of interest will include the following:. Randomized controlled trials and quasi-randomized trialsStudy designTiming. Studies with a minimum follow-up duration of 6 months after the first interventionLanguage. English languageSetting. Any settingWhile we have structured interventions into broad categories , our primary analyses will consider interventions at a more granular level to ensure findings are of maximal clinical relevance. We will pursue separate sets of analyses relating to pharmacologic and surgical interventions, as this approach is in alignment with clinical decision-making. After study data has been extracted, we will consult our clinical experts to determine whether additional treatment nodes or other modifications related to network geometry are needed to enhance representativeness of interventions . If combination therapies are encountered, we will include them as additional treatment groups in the analyses to be performed.The search strategies will be developed and tested through an iterative process by an experienced medical information specialist in consultation with the review team. The strategies will be peer reviewed by another senior information specialist prior to execution using the PRESS Checklist  Using thStrategies will utilize a combination of controlled vocabulary  and keywords . Results will be filtered using headings for systematic reviews, randomized controlled trials, and non-randomized controlled trials as applicable for each database. Vocabulary and syntax will be adjusted across databases. There will be no language or date restrictions on any of the searches, but when possible, animal-only and opinion pieces will be removed from the results. Potentially relevant articles published in languages other than English will be included in an Additional file. We will document any unforeseen limitations pertaining to search strategy upon its completion.ClinicalTrials.gov, and the International Clinical Trials Registry Platform will also be undertaken. Studies included in existing reviews will be inspected to confirm no relevant studies have been missed. Content experts will be contacted to obtain information on unknown or ongoing studies. The proposed database search strategies are provided in Additional file A gray literature search of targeted clinical trial registries, Screening will be performed in two stages via two reviewers working independently and in duplicate against eligibility criteria established a priori, using an online systematic review software program (Distiller Systematic Review (DSR) Software; Evidence Partners Inc, Ottawa, Canada). Stage 1 screening will be based on review of the abstracts and titles identified from the electronic search, while stage 2 screening will consider full-text review of the articles deemed potentially relevant during stage 1. Screening at both stages will commence with a calibration exercise to ensure consistent application of eligibility criteria. A screening pilot will be performed prior to full screening of titles and abstracts (25 titles and abstracts) and full-text screening (25 studies). At stage 1, two reviewers (NA and LE) will independently assess the titles and abstracts for eligible studies using the liberal accelerated method  where onA standardized data extraction form in Microsoft Excel  will be used for collecting key study information that includes all pre-specified data items  initiative  but did We will use the Cochrane Risk of Bias Tool for RCTs  to evaluA.Criteria for quantitative synthesisTo assess the assumptions for conducting NMA , a variety of information related to study methods, patient demographics, and eligibility criteria will be collected. These will include the following: mean age at onset, percent female patients, mean disease duration, frequency/severity measures of vertigo at baseline, percent with history of migraine headaches, percent with previously identified tinnitus, measures of average tinnitus intensity, and other factors. These study features will be reviewed with our clinical experts using a combination of table summaries, box plots, and bar plots to identify potential outlier studies that may warrant exclusion from formal analyses.I2 statistic) if data permit.B.Planned quantitative analysisAs noted earlier, separate sets of analyses pertaining to comparison of pharmacologic interventions and surgical interventions will be performed. Initially, we will inspect the characteristics of included studies such as patients’ clinical characteristics  and methodologic homogeneity , and we will summarize them accordingly. A pairwise meta-analysis for each intervention comparison will be pursued to explore statistical heterogeneity , we will explore subgroup analyses , 33 to eIf excessive heterogeneity is identified and the research team feels meta-analysis is inappropriate, a narrative summary of findings with supporting tables and figures will be prepared.To date, there have been a variety of pharmaceuticals used for the treatment of MD, including systemic drugs and intra-tympanic medications as well as surgical approaches. There exist systematic reviews comparing effectiveness of pairs of interventions in patients with MD –38. Howewww.clinicaltrials.gov and other registries to establish the current state of the evidence. In keeping with the COMET initiative’s recommendation regarding the establishment of standards for outcome selection [We will publish the findings of our review in a clinical specialty journal to enhance outreach to clinicians pursuing prospective research on MD. We will report evidence networks collating completed studies as well as ongoing trials identified from election , this reAdditional file 1. PRISMA checklist.Additional file 2. Search strategy.Additional file 3. Data extraction form.


Insulin is a vascular hormone and regulates vascular tone and reactivity. Muscle is a major insulin target that is responsible for the majority of insulin-stimulated glucose use. Evidence confirms that muscle microvasculature is an important insulin action site and critically regulates insulin delivery to muscle and action on myocytes, thereby affecting insulin-mediated glucose disposal. Insulin via activation of its signaling cascade in the endothelial cells increases muscle microvascular perfusion, which leads to an expansion of the endothelial exchange surface area. Insulin’s microvascular actions closely couple with its metabolic actions in muscle and blockade of insulin-mediated microvascular perfusion reduces insulin-stimulated muscle glucose disposal. Type 2 diabetes is associated with chronic low-grade inflammation, which engenders both metabolic and microvascular insulin resistance through endocrine, autocrine and paracrine actions of multiple pro-inflammatory factors. Here, we review the crucial role of muscle microvasculature in the regulation of insulin action in muscle and how inflammation in the muscle microvasculature affects insulin’s microvascular actions as well as metabolic actions. We propose that microvascular insulin resistance induced by inflammation is an early event in the development of metabolic insulin resistance and eventually type 2 diabetes and its related cardiovascular complications, and thus is a potential therapeutic target for the prevention or treatment of obesity and diabetes. Diabetes prevalence continues to increase at an alarming rate and has become the most common and costly chronic disease worldwide. This pandemic currently affects over 400 million individuals, largely due to a rapid increase in the prevalence of type 2 diabetes mellitus (T2DM) . AlthougInsulin, in addition to exerting metabolic actions on traditional peripheral insulin response tissues such as liver, skeletal muscle and adipose tissue, actively regulates vascular tone and tissue perfusion and these actions have been linked to the pathogenesis of diabetes and its cardiovascular complications. Vascular endothelium, a single endothelial cell layer lining the inner surface of the vascular lumen, expresses abundant insulin receptors as well as the insulin-like growth factor I (IGF-1) receptors and the hybrid insulin/IGF-1 receptors ,15,16,17Insulin fine-tunes vascular tone and health via balancing its signals through these two signaling pathways in the basal state but, at high physiological levels, as seen during euglycemic hyperinsulinemic clamp or in the postprandial state, insulin’s vasodilatory effect predominates. Ample evidence has confirmed that insulin exerts a vasoactive action at all levels of the arterial tree. It acts on large conduit arteries to increase compliance ,24,25, tSkeletal muscle is one of the main targets of insulin and responsible for approximately 80% of insulin-mediated glucose uptake ,32. To aThe muscle microvascular endothelium layer is continuous without fenestration and has tight junctions between the endothelial cells. The endothelium layer forms a barrier to prevent muscle cells from being exposed to toxic metabolic wastes and harmful inflammatory factors as well as shield the myocytes from being overly stimulated by hormones, nutrients, and numerous other factors, including insulin. Studies using cultured endothelial cells showed that insulin itself also facilitates its own trans-endothelial transport which is dependent on its binding to the insulin receptor and the receptor activation as inhibition of insulin receptor signaling blunts this process ,58,59. TThe close coupling between insulin’s microvascular actions and metabolic actions in muscle is further supported by the observation that metabolic insulin resistance coexists with microvascular insulin resistance in both clinical and animal studies. In humans or animals with either chronic or acute  insulin resistance microvascular responses to insulin are unequivocally reduced ,65,66,67As muscle microvascular endothelial exchange surface area is important in the delivery of insulin to muscle interstitium and thus insulin action in muscle, one would expect that expansion of the microvascular blood volume would result in increased delivery of insulin to the muscle and thus enhance insulin-mediated muscle disposal in the insulin resistant states. Indeed, studies from our and other laboratories have shown repeatedly that expansion of muscle microvascular blood volume with various agents can partially prevent or even reverse insulin resistance in rodents. Administration of losartan acutely expands muscle microvascular blood volume and this effect is associated with increased muscle use of glucose in the post-absorptive state and a full restoration of muscle insulin sensitivity in rats receiving systemic lipid infusion ,50,51. STogether, these studies highlight an important link between microvascular and metabolic effects of insulin in muscle and indicate that a loss of microvascular insulin responses contributes to the development or worsening of metabolic insulin resistance and expansion of the muscle microvascular volume experimentally enhances metabolic insulin responses. As such, microvascular insulin resistance may play a pathogenesis role in the development of T2DM and a therapeutic target for the prevention and management of T2DM. Insulin resistance as seen in obesity and T2DM is associated with a chronic inflammatory state with increased tissue infiltration of immune cells in multiple organs and tissues such as adipose tissue, liver, skeletal muscle, pancreas, and brain, and people with or animal models of obesity and diabetes have elevated plasma levels of inflammatory mediators such as TNF-α, C-reactive protein, and FFAs ,77. WhilFirst, the circulating pro-inflammatory cytokines secreted and FFAs released from the adipose tissue, particularly the visceral fat depot, have been well studied and confirmed to cause insulin resistance in muscle ,79,80. TVascular inflammation is a well-recognized phenomenon in obesity and diabetes and contributes to both endothelial dysfunction and insulin resistance, which usually coexist, mutually perpetuate, and contribute to the development of metabolic insulin resistance and related cardiovascular complications. Vascular walls in the insulin resistant state are inflammatory and pro-atherogenic. Serving as a barrier to protect tissue cells with various biological functions from harmful metabolic wastes or products, endothelium is constantly exposed to high levels of circulating inflammatory cytokines, FFAs and insulin, which not only induce insulin resistance in endothelial cells but also significantly increase endothelial cell synthesis of pro-inflammatory factors and adhesion molecules. Insulin at physiological concentrations only selectively stimulates insulin receptors, while it activates insulin as well as IGF-1 signaling in endothelial cells at high concentrations, as seen in the insulin resistant states, and both insulin and IGF-1 receptors mediate insulin stimulated endothelial production of adhesion molecules and monocyte adhesion to the endothelial cells . Insulin-mediated microvascular recruitment depends on normal endothelial response to insulin and endothelial dysfunction negatively impacts insulin’s microvascular action thereby contributing to the development of vascular insulin resistance. Inflammation clearly induces microvascular insulin resistance and reduces endothelial NO availability. Pro-inflammatory cytokines increase endothelial production of ROS and cause oxidative stress, leading to increased endothelial generation of superoxide instead of NO, a condition known as uncoupling . ExcessiInflammation also decreases trans-endothelial insulin transport, a rate limiting step in muscle insulin action. Endothelial cells are able to actively take up insulin which is the first step of the trans-endothelial transport process and this is significantly reduced in the presence of TNF-α . FeedingIn addition to regulating blood flow thus insulin delivery to the muscle, NO also inhibits endothelial cellular senescence  and decrvs. obese women remains unclear, it is likely that the differences in adipocyte size and cytokine/adipokine profiles may have contributed.While both the endocrine effects of the circulating cytokines and FFAs and the paracrine effects of myokines from myocytes and cytokines and FFAs from muscle fat depots are important in microvascular inflammation and insulin resistance, the role of perivascular adipose tissue (PVAT), adipose tissues surrounding the small blood vessels, on microvascular function and insulin sensitivity must be emphasized . In receInsulin is a vasoactive hormone and regulates its own delivery to and action in muscle by relaxing pre-capillary arterioles to recruit microvasculature and facilitating its own trans-endothelial transport. Muscle microvasculature provides the needed endothelial surface area for substrate exchange and insulin transport thereby affecting insulin-mediated glucose disposal and is a key linkage between insulin’s vascular and metabolic actions. Insulin resistance is a low-grade chronic inflammation state and inflammation in the muscle microvasculature blunts insulin-mediated microvascular recruitment and reduces trans-endothelial insulin transport, thus reducing insulin delivery to and action in muscle. A schema of the interplay among muscle microvascular inflammation, insulin delivery and insulin resistance is shown in