We present a case of Non-Arteritic Anterior Ischemic Optic Neuropathy (NA-AION) with uncertain etiology but a good recovery with a total gain of central visual acuity. Ischemic optic neuropathy is an acute ischemic disorder of the optic nerve.1,2].It is the most common acute optic neuropathy in patients over 50 years old, with an estimated annual incidence in the United States of 2.3–10.2 per 100 000 population .Ischemic optic neuropathies are of two types: Posterior Ischemic Optic Neuropathy, caused by ischemia of the posterior segment of the optic nerve, vascularized by multiple vessels, and Anterior Ischemic Optic Neuropathy, caused by ischemia of the anterior part of the optic nerve, supplied by the short posterior ciliary artery circulation. Etiologically and pathogenetically, AION is of the following two types: arteritic AION (A-AION), which is due to giant cell arteritis (Horton disease), and nonarteritic AION (NA-AION), which is due to all other causes . NAION presents with loss of vision occurring over hours to days, often described as blurring, dimness, or cloudiness in the affected region of the visual field, most often inferiorly. NAION typically presents without pain, although some form of periocular discomfort is reported in 8–12% of affected individuals [7]. Color vision loss in NA-AION tends to parallel visual acuity loss, as opposed to that in optic neuritis, in which color loss is often disproportionately greater than visual acuity loss. Visual field defects in NA-AION may follow any pattern related to optic nerve damage, but altitudinal loss, usually inferior, occurs in the majority, ranging from 55 to 80% of the reported cases [7-9].Visual acuity in patients with NA-AION varies considerably, from 20/ 20 (logMAR 0) to no perception of light; however, in general, the drop in acuity in patients with NA-AION is less than that experienced by patients with the arteritic form of AION, with over 50% of the patients having acuity better than 20/ 200 (logMAR 1) .The prognosis of this case is reserved. Optic nerve atrophy is irreversible, and currently there is no effective treatment to help nerve regeneration [There are several theories to be discussed regarding the physiopathology of NA-AION. 15], however, the rare cases that have been studied histopathologically have shown no evidence of thrombosis of the SPCAs  and it is therefore believed that the condition is, in some cases, due to generalized hypoperfusion and in others to occlusion of disc or laminar capillaries. The lack of consistent choroidal filling delay in fluorescein angiography studies of NA-AION suggests that the impaired perfusion arises in the paraoptic tributaries of the SPCA’s, distal to their split from the choroidal branches [18].Blood flow through the short posterior ciliary arteries (SPCAs) is reduced in patients with NAION [19].Persistent hypoperfusion requires impairment in the normal autoregulatory mechanisms of the optic nerve head. Flow is normally maintained constant with variations in perfusion pressure, intraocular pressure, and metabolic conditions (including tissue oxygen and CO2 levels) by factors that vary resistance to flow, such as the autonomic input. These autoregulatory mechanisms may be impaired by arteriosclerosis, chronic arterial hypertension, vasospasm, or medications, including beta-blockers and other antihypertensive medications [20], but the mechanism by which this contributes to ischemia has not been completely elucidated, and the role of additional factors such as nocturnal hypotension and sleep apnoea is unproven [21-27].Structurally small, “crowded” optic discs are associated with NA-AION [20]. The structure of the optic disc as a relatively inflexible region encompassing the axons of the optic nerve has been implicated in two histopathologic studies suggesting a compartment syndrome as a mechanism . Almost all patients who develop NA-AION have a “disc-at-risk” [30].Almost all patients who develop NA-AION have at least one underlying vascular risk factor that may or may not be known at the time they lose vision. Nocturnal systemic hypotension may have a significant role in the development of NA-AION, particularly in patients with an exaggerated nocturnal “dip” or in patients, such as those with systemic hypertension, in whom optic disc circulation autoregulatory mechanisms are impaired [31-33], hyperlipidaemia . Smoking is also a significant risk factor on the basis that smokers developed NA-AION at a significantly younger age than nonsmokers [36]. NAION has been reported in association with many conditions that may predispose to decreased optic nerve head perfusion via microvascular occlusion: systemic hypertension, diabetes mellitus [37-40].Several medications have been associated with the development of NA-AION including Interferon-alpha via systemic hypotension or immune complex deposition within the optic disc circulation. Three drugs that inhibit phosphodiesterase 5—sildenafil (Viagra), tadalafil , and vardenafil (Levitra) may produce systemic hypotension. Amiodarone is in widespread use as a cardiac anti-arrhythmic agent and has been associated with the development of an anterior optic neuropathy that clinically mimics NA-AION . Secondary neuronal degeneration in cells adjacent to infarcted tissue may develop as a result of a toxic environment produced by the dying cells [43]. Such deterioration may be mediated by processes including excitatory amino acid  toxicity, reactive oxygen species (including lipid peroxidation), intracellular calcium influx, and apoptosis . Ischaemia-induced cell death may result in release of glutamate, with further cell damage and death by excitotoxic induction of apoptosis . Unique animal models for optic nerve ischaemia bring fluorescein angiography based and electrophysiological evidence of early optic nerve axon damage and delayed retinal ganglion cell loss [46].Concerning the cellular mechanisms of the NA-AION, recent advances in the understanding of ischaemic central nervous system damage have raised new questions regarding the pathogenesis of neuronal damage in both the arteritic and nonarteritic forms of AION. Neurotrophin deprivation in retinal ganglion cells after ischaemic insult may have a significant role in cell death .Interestingly, histological and immunohistochemical assessments, indicate a significant inflammatory reaction in the region of the ischaemic lesion, suggesting that at least some of the optic nerve damage is related to inflammation rather than ischaemia [Several therapeutic approaches have been tried over the years and we are trying to review and comment them.49]. The presumed mechanism through which corticosteroids improve the outcome in NA-AION patients is prevention of the “vicious circle” [50] in which the ischemic tissue further suffers from the secondary damage by a mechanical pressure caused by the swollen ischemic axons in an already crowded disc with a small scleral canal. This would not prevent the primary insult but should theoretically limit the secondary insult. Reducing capillary permeability in the optic disc [51] could be another mechanism.Mega-dose intravenous corticosteroid treatment was also proposed as a treatment for NA-AION [52], and the mechanical damage caused by the swelling itself, a boost of IV corticosteroids  would be more efficient, as was found for optic neuritis in the ONTT [53].It is feasible that if the optic nerve could be “saved” from the secondary damage caused by inflammation . Other studies revealed no difference in visual outcome between treated and untreated subjects [55]. Systemic oral corticosteroid administration showed some evidence of improving visual function compared to the natural evolution of acute NA-AION [56], and can be administered intravenously, intravitreally [57], or even topically. Erythropoietin has been shown to be neuroprotective in vitro and in animal models of neuronal injury [58].Hyperbaric oxygen was proven to have no beneficial effect .The IONDT  assessed the value of optic nerve sheath fenestration (ONSF) in the treatment of acute NA-AION, based on the theory that at least some of the optic nerve damage caused by NA-AION in which there is progressive visual loss is due to post-ischaemic intraneural swelling with secondary impairment of local vascular flow or axoplasmic transport within the optic nerve head and that reduction of perineural subarachnoid cerebrospinal fluid pressure could improve vascular, axonal transport, or both, thus reducing tissue injury in reversibly damaged axons. However, data analysis revealed no significant benefit of this type of treatment [61]. However, this is a rather invasive approach. Transvitreal optic neurotomy has been proposed as a therapy. If a compartment syndrome is at least a component of the pathophysiology of NA-AION, in theory, such a procedure could break the cycle of edema and vascular compression , vasodilators [6], heparin-induced extracorporeal LDL/ fibrinogen precipitation (HELP) [8], hyperbaric oxygen [9], diphenylhydantoin [62], norepinephrine [63], levodopa [64], topical brimonidine  and intravitreal bevacizumab [20].Other studies explored the role of aspirin , others [69] found no long-term benefit for aspirin use. Nevertheless, although beneficial long-term effects remain unproven for NA-AION, many experts recommend the use of aspirin after an initial episode, only for its role in decreasing risk for stroke and myocardial infarction in this vasculopathy population group.Just as there is no proven treatment for NA-AION, there is no proven prophylaxis to prevent second-eye involvement. Although some authors have found evidence that aspirin can reduce the incidence of fellow-eye involvement after NA-AION .NA-AION has a probability of only 5% to relapse in the same eye. There is a risk of congener eye damage: 25% at 3 years, 17% at 5 years and 15% at over 5 years [Particular for this case was that the patient arrived in our clinic at the onset of the symptomatology, and there was a rapid central visual acuity gain, 20/ 20 at 3 weeks, even though there is no efficient treatment demonstrated for NA-AION in the specialty literature. The exact cause of the papillary edema has yet to be determined, with no history of hypovolemia, with a drug controlled arterial hypertension and a clinical and paraclinical examination of other systems and organs within normal limits. This causes frustration for both the doctor and the patient.However, the physical effort and the event with emotional impact, associated with anxiety and arterial hypertension, may have contributed to a hypertensive jump that precipitated the start of NA-AION.The burden of the treating physician is the chase “towards the end of the tunnel” for the widening of the central area of sight with limited therapeutic resources.Financial DisclosuresNone of the other authors has any financial or proprietary interests to disclose.


The historical lack of models recapitulating the complexity of the human intestinal epithelium has hindered studies into many aspects of human enteric virus biology. Immortalized and transformed cell lines are typically limited by the presence of only one cell type, whereas susceptibility in animal models often requires infection routes that differ from humans or necessitates modification of immune system components , 2. In aGastrointestinal organoids are three-dimensional 3D) structures derived from primary tissues  containing intestinal stem/progenitor cells or from human pluripotent stem cells (hPSCs) D structu and may Fig 1). Future studies will determine whether they also provide an avenue for the cultivation of other currently nonculturable human enteric viruses such as sapoviruses, picobirnaviruses, and some picornaviruses , as well as other not previously examined genogroups of the astroviruses (VA2-5 and MLB2-3).HIOs and HIEs are currently being used to study host–virus interactions and have been successfully implemented for difficult-to-cultivate enteric viruses. Enteric viruses in clinical samples have been traditionally refractive to replication in transformed cell lines . HoweverFig 1) pointing to a mechanism of diarrhea induction, the infection of other cell types is likewise critical for viral pathogenesis. For example, infection of enteroendocrine cells provides an explanation for induction of vomiting responses in the host [Multiple cell types contribute to the complexity of intestinal epithelium and its many functions. Single cell and bulk RNA sequencing, flow cytometry, and immunofluorescence data have demonstrated that HIOs and HIEs possess distinct epithelial cell types, highlighted by cell-specific markers including mucin-2 (goblet), Lgr5 (stem), E-cadherin, sucrase isomaltase (enterocytes), Lysozyme-C (Paneth), and chromogranin A (enteroendocrine) cells , 19, 23.the host , whereasthe host  and mucothe host .Another advantage of the HIO/HIE model is their nontransformed status, thus enabling studies into the human host response to viral infection. In particular, many transformed cell lines have defects in innate immune signaling , confounThe ability to study clinical isolates that do not grow in transformed cells represents one application of HIOs/HIEs toward the identification of antiviral strategies. For example, HIEs were used to demonstrate the attenuated infectivity of a rotavirus vaccine strain . HIEs alHIEs are also used to study pathophysiological responses to enteric virus infection, such as demonstrating diarrhea with fluid section in rotavirus-infected HIEs . GeneticThe advent of gastrointestinal organoid technology has been a critical breakthrough for the study of virus–host interactions. For enteric viruses, the use of this technology to develop cell culture systems for uncultivable enteric viruses has been transformative. Key findings have already been made regarding their human cell tropism, effectiveness of antivirals and disinfectants, and human antiviral innate immune responses and viral counteraction of these. However, to date, these are generally individual findings for a particular virus family. The broad application of this technology across the enteric virus families has the potential to lead to additional discoveries in enteric virus biology and the identification of common principles of these viruses. Furthermore, additional modification of the intestinal organoid models to ever more closely mimic the human intestine in a dish offers unprecedented opportunities for future discovery.


Many different receptor systems are regulated by endomembrane traffic, including signals from receptors activated by extracellular cues such as receptor tyrosine kinases (RTKs), G protein-coupled receptors (GPCRs), morphogen receptors, and immune receptors. Notably, with few exceptions, each receptor system undergoes internalization from the cell surface upon activation, followed by receptor-specific control of intracellular membrane traffic through complex intracellular endosomal compartments. The canonical view of regulation of receptor signaling by endocytosis involves downregulation of receptors by their sequestration from the extracellular milieu rich in ligands or degradation of receptor-ligand complexes following traffic to the lysosome. However, it has become apparent that a wide variety of receptors and their downstream signals exhibit actions unique to specific endomembrane compartments , a phenomenon which may require endosomal recruitment of receptor and signaling intermediates. In addition, the potential for pharmacological intervention of putative endosomal signals activated by PAR1 and other GPCRs that trigger p38 MAPK activation for treatment of patients with intracranial hemorrhage and other injuries is highlighted.The role of ubiquitinylation in promoting the lysosomal degradation of signaling receptors has been well-documented. Focusing on GPCRs, Lin and Liu reported that mTOR in skeletal muscle is recruited to and activated at macropinosomes. Mechanical stretch and osmotic shock induced activation of phospholipase D2, but not D1, and stimulated phosphatidic acid production on macropinosomes, leading to mTOR signaling. The surprising finding that mTOR can be activated in macropinosomes, in addition to lysosomes, underscores the complexity of the spatiotemporal dynamics of mTOR signaling that has implication in development and disease progression.mTOR (mechanistic target of rapamycin) signaling that regulates cell growth and proliferation has been classically associated with the lysosomes. Under mechanical stimulation, Inapanathan and Botelho examine how lysosomes serve as signaling hubs, functioning as platforms for recruitment and regulation of important signaling proteins such as AMP-activated protein kinase (AMPK), mTORC complex 1 (mTORC1), and glycogen synthase kinase 3ß (GSK3ß). This review examines how the highly regulated localization of each of these signals to the surface of the lysosome allows integration of cues such as from cell metabolism, extracellular mitogens, and immune signals into a coordinated cellular response. The review further discusses how these lysosome-derived signals in turn control autophagy, lysosome adaptation, cell proliferation and survival and immune cell function and many other outcomes, and how these observations support the view of lysosomes as a major signaling hub of the endocytic system.Lysosomes serve the critical function of effecting degradation of macromolecules within cells; however, these organelles are becoming well-appreciated as having far more complex functions that impact many aspects of cellular physiology. Onnis and Baldari reviewed a body of literature that points to trafficking pathways that deliver endosomal T-cell antigen receptor (TCRs), kinases, and adapter proteins to the IS in order to sustain signaling. TCRs can be internalized by both clathrin-dependent or clathrin-independent pathways, in a ligand-dependent or independent manner. Although classically known for signal termination, TCR endocytosis is followed by distinct polarized recycling pathways that contributes to IS assembly. Thus, paradoxically, TCR endocytosis is important for both signal termination and sustained signaling. The exocytic trafficking at the IS is also believed to be involved in intercellular communication to modulate antigen presenting cell function.The interplay between plasma membrane organization and vesicular trafficking and signaling is nicely illustrated in the context of immunological synapse (IS), a specialized contact area between a T cell and an antigen presenting cell. Sugiyama et al., we examine the compartmentalization of PI3K and Akt activation, regulation and signaling to downstream substrates on various membrane compartments including the plasma membrane, endosomes, lysosomes, and mitochondria, as well as within nanoscale domains within compartments such as the plasma membrane. A particular focus is the recruitment of PI3K-Akt signals and regulators to the plasma membrane and endocytic vesicles derived therefrom, as this may suggest a mechanism for spatiotemporal coordination of positive and negative regulators of this signaling axis. Furthermore, this review examines how the compartmentalization of these key signals at various scales within the endocytic network impacts cellular processes such as invadopodia formation, metabolism, and lysosomal function.The endocytic system is intimately linked with signaling from RTKs, in particular for signals involving phosphatidylinositol-3-kinase (PI3K) and Akt. In DeNies et al., we investigated the relationship between clathrin-mediated receptor internalization and signaling for a chemokine receptor CXCR4. As expected, CXCR4 internalization is significantly reduced when clathrin is knocked down. Interestingly, we reported an increase in chemokine CXCL12-dependent ERK1/2 signaling but a decrease in cell migration under clathrin-knock down condition. However, disrupting lipid raft and CME simultaneously abrogated ERK1/2 signaling. An antibody sensitive to CXCR4 post-translational modification (PTM) revealed a decrease in CXCR4 PTM upon clathrin knock down, despite an unchanged total CXCR4 protein and mRNA levels. The study highlights the growing understanding that modulation of endocytosis affects not only receptor signaling and internalization but also receptor PTM.The interplay between receptor internalization and signaling is expected to take place during directed cell migration process such chemotaxis. In Altogether, these studies and topical reviews underline the importance and regulation of signal compartmentalization at different cellular locales. Important unanswered questions remain, such as exactly how intracellular pools of receptors communicate with cell surface activated receptors and extracellular ligands. Could internal receptors be post-translationally modified in the absence of ligand stimulation? If so, what might be the physiological relevance of such signal compartmentalization? What are the unique physiochemical properties of specific endocytic compartments that endow these with unique signaling factors, and what is the specific repertoire of signaling factors in each endocytic compartment? Systems biology of receptor trafficking and signaling approaches will be important to unravel the mystery of spatial compartmentalization of receptor signaling. Finally, how receptor crosstalk plays a role in signaling compartmentalization, and how alterations of endocytic traffic can contribute to specific diseases by driving changes in receptor signaling will also be important future research directions.CA and AL both conceived and wrote the editorial.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.


Age is a primary risk factor for a number of chronic diseases including mobility disability, cardiovascular disease (CVD), type 2 diabetes (T2D), and cancer. Most physical activity guidelines emphasize the performance of 150 min of moderate-to-vigorous or 75 min of vigorous aerobic exercise training (AET) weekly for reduction of chronic disease risk. Nonetheless, there is an emerging body of evidence showing that resistance exercise training (RET) appears to be as effective as AET in reducing risk of several chronic diseases. It may also be that RET is more effective than AET in some regards; the converse is likely also true. We posit that the perceived divergent exercise mode-dependent health benefits of AET and RET are likely small in most cases. In this short review, our aim is to examine evidence of associations between the performance of RET and chronic health disease risk . We also postulate on how RET may be influencing chronic disease risk and how it is a critical component for healthy aging. Accumulating evidence points to RET as a potent and robust preventive strategy against a number of chronic diseases traditionally associated with the performance of AET, but evidence favors RET as a potent countermeasure against declines in mobility. On the basis of this review we propose that the promotion of RET should assume a more prominent position in exercise guidelines particularly for older persons. Cardiovascular disease (CVD), cancer, and type 2 diabetes (T2D) are leading causes of morbidity and mortality in older adults aged 65 years and older . Aging iThe progressive decline of skeletal muscle mass and strength with aging is collectively referred to as sarcopenia, and is prognostic for mobility disability , 2005 anResistance exercise (RE) and aerobic exercise (AE) are modalities of exercise that are traditionally conceptualized as existing on opposite ends of an exercise continuum in terms of the phenotypes they lead to. A common misconception is that RE training (RET) and AE training (AET) also result in separate health benefits, but we propose this is an artifact of the greater volume of data that currently exists for AET as opposed to RET. Currently, most physical activity guidelines advise, as their primary message, that older adults should perform at least 150 min of moderate-to-vigorous or 75 min of vigorous AET weekly for the reduction of chronic disease risk and maintenance of functional abilities . HoweverThe aim of this review is to provide an up-to-date evidence-based narrative review of the efficacy of RET in combating chronic health disease  risk in older adults. To achieve this aim, we summarize data derived predominantly from humans, but will draw upon important findings from preclinical disease models to substantiate our arguments and provide additional mechanistic insight not available in human observational trials.Mounting evidence from systematic reviews , meta-anA recent umbrella review demonstrated that RET in pre-frail and frail older adults could significantly enhance muscular strength, gait speed, and physical performance . Pooled peak]; The underlying mechanisms by which RET attenuates the decline in physical function of older adults is likely multifaceted. However, low muscle mass and strength are associated with poor physical function , and preAlthough high-intensity RET (≥70% of 1RM) is generally more effective than low-to-moderate intensity RET (30–69% of 1RM) in combating mobility decrements , the hetA hallmark of aging is the progressive deterioration of whole-body insulin sensitivity and consequent impairment of glycemic control  that preGiven that ~80% of glucose is deposited in skeletal muscle during postprandial periods , the losRecent work from our laboratory demonstrated that a reduction of habitual daily stepping for a period of 2 weeks  in prediabetic older adults results in significant impairments in glycemic control and an insulin-resistant state in response to a 75-g oral glucose challenge . ImportaLifestyle interventions are arguably the most effective therapeutic strategies in terms of preventing and managing diabetes. Indeed, the Diabetes Prevention Program (DPP) demonstrated that lifestyle modifications  were associated with a greater reduction  or whole-body RE (3 sets at 70% 1RM) resulted in significant reductions of plasma glucose levels in physically active type 1 diabetics  exerts similar improvements in blood pressure , and bloCancer is a leading cause of morbidity and mortality with approximately 14 million new cases and 9.6 million annual cancer-related deaths worldwide . Many ofUsing data derived from the Health Survey for England and the Scottish Health Survey consisting of 80,000 adults aged >30 years, 2max) during radiation therapy (2max) and usual care  for cancer exacerbate the loss of skeletal muscle mass and strength in patients. Importantly, these adaptions have negative implications for vital clinical endpoints including cancer mortality , disease therapy . Excitinual care . In a reual care . These fual care . Furtherual care .Several biological mechanisms have been proposed to mediate the protective effects of RET on cancer risk and prognosis. RET improves indices of insulin sensitivity, body composition , immune Similar to AET, there is a role for RET in reducing cancer risk, cancer recurrence, cancer mortality, and improving prognosis during adjuvant therapies. Given that cancer has surpassed CVD as the leading cause of death in several developed countries , these oThe wide-ranging health benefits of regular RET are well established; however, adherence to RET in older adults remains low, and the most commonly cited barriers to participation of RET are: (1) risk of injury (from lifting heavy relative loads) and (2) required access to a gym facility . However“another tool in the toolbox” for older adults to remain physically active and combat chronic disease risk. We do acknowledge that some knowledge gaps exist such as the optimal dose and intensity of RET required to exert health benefits and clinical trial evidence showing head-to-head comparisons with AET, and further investigations are needed.The evidence presented in this review demonstrates the beneficial effects of RET on reducing chronic disease risk  in older adults . RegularJCM, TS, and SMP wrote the initial draft of the manuscript. All authors edited and approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.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.


When it comes to the epigenome, there is a fine line between clarity and confusion—walk that line and you will discover another fascinating level of transcription control. With the genetic code representing the cornerstone of rules for information that is encoded to proteins somewhere above the genome level there is a set of rules by which chemical information is also read. These epigenetic modifications show a different side of the genetic code that is diverse and regulated, hence modifying genetic transcription transiently, ranging from short- to long-term alterations. While this complexity brings exquisite control it also poses a formidable challenge to efforts to decode mechanisms underlying complex disease. Recent technological and computational advances have improved unbiased acquisition of epigenomic patterns to improve our understanding of the complex chromatin landscape. Key to resolving distinct chromatin signatures of diabetic complications is the identification of the true physiological targets of regulatory proteins, such as reader proteins that recognise, writer proteins that deposit and eraser proteins that remove specific chemical moieties. But how might a diverse group of proteins regulate the diabetic landscape from an epigenomic perspective? Drawing from an ever-expanding compendium of experimental and clinical studies, this review details the current state-of-play and provides a perspective of chromatin-dependent mechanisms implicated in diabetic complications, with a special focus on diabetic nephropathy. We hypothesise a codified signature of the diabetic epigenome and provide examples of prime candidates for chemical modification. As for the pharmacological control of epigenetic marks, we explore future strategies to expedite and refine the search for clinically relevant discoveries. We also consider the challenges associated with therapeutic strategies targeting epigenetic pathways.The online version of this article (10.1007/s00125-017-4490-1) contains a slideset of the figures for download, which is available to authorised users. Vascular disease affecting nearly all types of blood vessels is common to both type 1 and type 2 diabetes mellitus. Accelerated rates of clinically defined macrovascular complications, such as myocardial infarction and stroke, which result from large vessel atherosclerosis remain the leading causes of morbidity and premature mortality in the diabetic population. Diabetes is also associated with the occurrence of adverse microvascular complications, manifesting clinically as retinopathy, neuropathy and nephropathy.Extensive debate surrounds the extent to which diabetic microvascular and macrovascular complications represent a continuous pathological spectrum. Closely related to this debate is the question of why not all people with diabetes complications experience more advanced forms of vascular disease. Though yet to be completely defined mechanistically, the persistent and harmful effects of antecedent hyperglycaemia may at least partly explain the variation in vascular deterioration. Nonetheless, the fundamental reasons why a proportion of diabetic individuals appear to be protected from serious complications remain poorly understood. Despite the promises of the genetic revolution, contemporary knowledge of the impact of genetic variation on diabetes does not adequately explain the disproportionate distribution and severity of diabetic vascular complications.Realisation of novel preventative and therapeutic approaches hinges on improved characterisation of the molecular events and interactions that underlie the development and progression of diabetic vasculopathology. Interestingly, however, some in the field have shifted their research focus to understanding the post-translational and covalent chemical chromatin modifications that contribute to transcriptional regulation via structural adaptation. Insight from cultured cells and preclinical models, as well as clinical samples, has highlighted the importance of chromatin modifications in the persistent inflammatory response to glycaemic variability. Thus, epigenetics may be able to provide an explanation as to why some individuals with diabetes are predisposed to developing vascular disease and are more likely to progress to advanced stages of complications and/or develop other associated vascular pathologies.As mentioned above, long-term inadequate glycaemic control is a major risk factor in the development of vascular complications. Despite the proclivity for patients with good metabolic control to have a significantly decreased risk for developing complications, vascular disease may still develop and progress even with intensive treatment regimens . This isThe landmark Diabetes Control and Complications Trial (DCCT) was the first to demonstrate that achieving near-normal blood glucose levels ameliorates microvascular complications of type 1 diabetes . MoreoveAlthough hyperglycaemia is demonstrably a principal cause of microvasculopathy—the microvasculature of the retina is particularly susceptible to excess glucose and diabetic nephropathy is not observed in the absence of hyperglycaemia —the effiUnlike the near inexorable progression to retinopathy, more than half of all individuals with type 1 diabetes do not develop renal complications . While iThus, the search for genetic factors associated with diabetic nephropathy susceptibility, using initially linkage analyses, candidate gene-based approaches, and, more recently, hypothesis-free GWAS has been extensive. Yet, even with the advent of modern sequencing technologies, intensive efforts have yielded only a limited number of consistent genetic associations, and the impact on clinical management has so far been negligible . Large cMethylation is unique in the way that it is enriched at cytosine bases of the DNA template, primarily, but not exclusively, at cytosine–guanine (CpG) dinucleotides, as well as on the tails of chromatinised histones. When written by DNA methyltransferase enzymes  to the 5-carbon position of cytosine , the methyl modification is historically associated with transcriptional silencing by recruitment of specific factors that actively remodel the chromatin structure, as well as by the disruption of transcription factor binding sites. On the other hand, 5mC enrichment can preclude binding of transcriptional repressors such as CCCTC-binding factor (CTCF), which is associated with altered chromatin structures and thus aberrant gene activation . Indeed,Methylation of lysine and arginine residues on histone tails is similarly associated with both transcriptional activation and repression, depending not only on the position of the substrate residue within a specified histone tail, but also the degree of modification  and NPHS2 . Structural disturbance of the slit diaphragm proteins results in insufficient filtration and proteinuria, exemplified by congenital kidney failure arising from defects in nephrin . PodocytKLF4) [NPHS1 expression and inversely with proteinuria in rodent and human podocytes [NPHS1 promoter and upregulates gene expression by reduced methylation or demethylation of adjacent CpG sites. Methyl profiling of cultured human podocytes overexpressing KLF4 revealed widespread changes in 5mC, highlighted by reduced CpG methylation at promoters of other epithelial genes, such as NPHS2 and SYNPO (which encodes synaptopodin), in striking contrast to hypermethylation at promoters of mesenchymal genes, such as VIM (which encodes vimentin) and CTGF (which encodes connective tissue growth factor), thereby indicating KLF4-dependent determination of podocyte phenotype by gene-specific methylation. The potential implications of these findings are significant, not only for slit diaphragm maintenance but also for epithelial to mesenchymal transition observed in advanced diabetic nephropathy. The slit diaphragm proteins NPHS1 and nephrin-like 3 (NEPH3) are encoded by genes (located on chromosome 19q13.12 in a head-to-head orientation) that share a bidirectional promoter and have been shown to be dependent on 5mC for co-regulation and expression [Expression of the pluripotency-associated Kruppel-like factor 4 (KLF4)  correlatodocytes . Moreovepression .TXNIP). Inhibition of EZH2 augments proteinuria, podocytopathy, glomerular Txnip expression, and renal oxidative stress in a rat model of diabetes [Txnip expression is coordinated by histone acetylation and methylation in kidneys from diabetic Sur1-E1506K+/+ mice [Shc in podocytes exposed to high glucose [Shc expression beyond restoration of euglycaemia in cultured vascular endothelial cells and diabetic mice [Shc gene regulation was further underscored by pharmacological and molecular experiments targeting class III HDACs, and specifically the NAD+-dependent SIRT1 HDAC [Specifically within the diabetic setting, the chromatin-dependent regulation of glucose-mediated oxidative stress in podocytes is emerging as a critical mediator of diabetic nephropathy. For example, the H3K27-methylating function of enhancer of zeste 2 repressive complex 2 subunit (EZH2) recently emerged as an important regulator of diabetic nephropathy by repressing the transcription factor PAX6 and subsequently dampening expression of the endogenous antioxidant inhibitor thioredoxin-interacting protein  and G6pc, indicating that 5mC underlies selective glucose handling by PTECs in the kidney [db/db mice exhibited a comparable genome-wide methylation profile to PTECs from control animals, significant differences in 5mC were observed at genes implicated in sugar reabsorption (Slc5a2), nephropathy (Met) and hypertension (Agt) [Agt was shown to accumulate aberrant epigenetic changes early in the pathogenesis of diabetic nephropathy. Acetylated H3K9 was enriched at the Agt promoter as early as 5 weeks in the diabetic kidney and preceded both DNA hypomethylation and H3K9m3. The importance of H3 acetylation was further underscored by Agt transcriptional activation in response to HDAC inhibition. In contrast, these epigenetic changes were resistant to the glucose-lowering drug pioglitazone. In human diabetic nephropathy, microdissected tubuli from individuals with diabetic kidney disease exhibited differential methylation of genes implicated in fibrogenesis [db/db mice [RAGE , PAI1  and MCP-1  promoters.Contiguous with podocytes in renal structure, proximal tubular epithelial cells (PTECs) play a crucial role in renal function, reabsorbing much of the glucose and amino acids, as well as sodium, from the glomerular filtrate. Both high glucose and abnormal protein trafficking through the glomerulus induce inflammation and tubulointerstitial lesions through PTEC activation, and the extent of interstitial fibrosis ultimately determines the rate of decline in renal function . Recent e kidney . While Pon (Agt) . An impoogenesis . Central/db mice . In mesaKidney biopsies from experimental diabetes models or individuals with diabetes are characterised by enhanced macrophage infiltration , 50. FurImmunological memory had classically been viewed as being characteristic of only the adaptive immune system (T and B lymphocytes). However, it has recently emerged that innate immune cells exhibit memory-like behaviour, characterised by an increased proinflammatory response to secondary infections –57. ImpoThe non-specific trained memory of monocytes is thought to have beneficial effects in numerous immunological settings, including vaccination programmes . In contChronic hyperglycaemia associates with monocyte activation, induced directly by glucose or by other endogenous compounds associated with hyperglycaemia, such as AGEs , 67. WheThe epigenetic reprogramming of monocytes clearly involves marked changes in cellular metabolism, which is determined by their activation status , 69. SpeInterestingly, the metabolic state of the cell is linked to a particular epigenetic program . Indeed,The relationship between metabolic processes and epigenetic changes has so far been studied predominantly in the field of cancer research , but couThe heritability of epigenomic signatures continues to be intensely debated, though several plausible mechanisms of transgenerational transmission of acquired phenotypes have been described (reviewed elsewhere ), includUNC13B), which is associated with glucose-mediated apoptosis in glomerular cells [Expansion of high-throughput sequencing technologies to include the profiling of chromatin modifications on a genome-wide scale represents a new approach towards understanding predisposition to diabetic nephropathy. Studies comparing diabetic individuals with and without diabetic nephropathy reported differential DNA methylation at numerous genes, including several previously identified by GWAS , 79, 80.ar cells , and is Immune cell subtypes are distinguished by epigenetic profiles , and difWhile certainly an attractive approach to understanding complex disease phenotypes, several challenges limit the interpretability of epigenomic profiles. Notably, the aforementioned studies have used methylation array hybridisation technology that predominantly assays promoters, and to date there are no unbiased profiles of DNA methylation in the context of diabetic nephropathy. Genome coverage is critical to understand differential methylation outside promoter regions. For example, the application of next-generation sequencing generates comprehensive maps of DNA methylation data, thereby reducing the limitations often attributed to array composition . ContrarAltered DNA methylation patterns at specific loci can distinguish phenotypic cases from controls to reveal possible causal mechanisms. However, the cross-sectional approach can also be confounded by reverse causation, where the interrogated epigenomes are shaped by (rather than cause) the disease , as descTXNIP, an effect that was replicated in a cell culture model of hyperglycaemic variability [Inter-individual epigenetic differences may prove to be valuable predictive biomarkers of diabetic nephropathy susceptibility and development. Progression of atherosclerosis can indeed be associated with the degree of DNA methylation within plaques , and proiability .As discussed previously, 5mC is a powerful regulatory determinant, and given that 60–90% of the cytosines in the adult vertebrate cell contain CpG methylation, the prospect that many other functional sites within the genome are potentially altered would seem highly likely, although there is limited experimental evidence of this Fig. . The mosWith increased exploration of chromatin modifications in various medical contexts, it may be possible in the future not only to track causal mechanisms of vascular disease, but also to capture an individual patient’s position within a complex spectrum of pathophysiological processes, thereby supporting tailored approaches to the anticipation and prevention of diabetic complications . The epiThe emerging picture of epigenetic regulation is one of remarkable complexity. Defining the tissue-specific relative contributions of epigenetic writers and erasers remains an unresolved but critical issue for developing novel therapeutic epigenetic modulators. Whereas HDAC3 deletion from the macrophage is vasculo-protective , deletioDespite these formidable challenges, the epigenome is rich in opportunity. Compounds that target epigenetic pathways are increasingly investigated pre-clinically, and drugs that are already used in clinical management of diabetes may impact the epigenetic landscape. For example, metformin prevents trained immunity by the Bacillus Calmette–Guérin (BCG) vaccine via mTOR inhibition and suppression of glycolysis . WhetherIncreasingly accessible technologies that permit unbiased acquisition of genome-wide patterns boast the capacity to transform our understanding of the chromatin landscape in the occurrence and progression of complex diseases. The immense potential for epigenetics to explain many aspects of diabetic vascular complications is evident in recent scientific literature. By sensitising the genome to environmental variation, these molecular signatures shape diverse phenotypes and functional programs. Chromatin modifications influence deleterious changes in gene expression that, under some circumstances, can endure improvements in metabolic management. Similarly, persistent epigenetic modifications drive the non-specific memory of proinflammatory macrophages, a process that is increasingly implicated in vascular disease and could prove to be instrumental to resolving the important debate as to whether diabetic microvascular and macrovascular pathology share a common pathology. Epigenomic profiling of circulating cells may further shed light on the phenotypic variation and disproportionate burden of diabetic vascular complications. Combined with classical genetic approaches, epigenomic profiling has potential to identify molecular trajectories underlying diabetic vascular disease development. While the extent that pathological chromatin changes can be manipulated in human diabetic complications remains to be established, the clinical applicability of epigenetic interventions will be greatly advanced by a deeper understanding of the cell type-specific functions and interactions of chromatin-modifying machinery in the diabetic vasculature.ESM Downloadable slideset(PPTX 771 kb)


Connexins (Cxs) and pannexins (Panxs) are ubiquitous membrane channel forming proteins that are critically involved in many aspects of vascular physiology and pathology. The permeation of ions and small metabolites through Panx channels, Cx hemichannels and gap junction channels confers a crucial role to these proteins in intercellular communication and in maintaining tissue homeostasis. This review provides an overview of current knowledge with respect to the pathophysiological role of these channels in large arteries, the microcirculation, veins, the lymphatic system and platelet function. The essential nature of these membrane proteins in vascular homeostasis is further emphasized by the pathologies that are linked to mutations and polymorphisms in Cx and Panx genes. The blood contains components, such as coagulation factors and platelets, that are essential to keeping the cardiovascular circuit closed after injury by initiating haemostasis and formation of a platelet clot. The systemic circulation is composed of large elastic arteries such as the aorta, serving as high-pressure conduits for the blood to smaller muscular arteries and arterioles. Arterioles are resistance arteries controlling blood flow into capillary beds by their high vasodilatory and vasoconstrictive ability. In the capillary beds, the actual exchange of O2/CO2, nutriments, catabolites and fluid takes place between the blood and the surrounding tissue. The blood returns to the heart via venules and veins. The excess of interstitial fluid returns to the systemic circulation via lymphatics, a blind-ended system of lymphatic capillaries converging into collecting vessels and ending into the subclavian vein. Much evidence has demonstrated an important role for connexins (Cxs) and pannexins (Panxs) in many aspects of vascular physiology and pathology. In this review, we will focus on the role of Cxs and Panxs in the physiology/pathophysiology of the vascular system by systematically following the route of the blood from the left ventricle through the systemic circulation to its way back to heart.The cardiovascular system consists of the heart pumping the blood to a closed circuit of interconnected blood vessels, allowing for the indispensable and constant supply of OGJA4). The names of Cx proteins, on the other hand, are determined by their specific molecular weight in kDa . Structurally, Cxs comprise 4 α-helical transmembrane domains (TM1–TM4) and two extracellular loops (EL1 and EL2) that are highly conserved among the family members. Substantial differences among Cxs, both in length and composition, are found in their cytoplasmic amino-terminal (NT) and carboxy-terminal (CT) parts, as well as in the intracellular loop (IL). The synthesis of Cxs occurs in the endoplasmic reticulum (ER) and their oligomerization in the ER/Golgi or trans-Golgi network results in the formation of hexameric connexons )The mechanism of α1-AR activation of Panx1 is still under investigation, although based on previous work it is thought that Panx1 may be selectively regulated by receptor stimulation at the intracellular loop . The useThere are other more specific regions where Panx1 may play a role in the microcirculation. There is no identified role yet for Panx1 in regulation of endothelial-mediated dilation, except in large conduit vessels, which do have augmented responses to endothelial-induced vasodilation in global Panx1 knockout animals . HoweverPanx1 utilization could also be considered vascular bed specific. For example, it has recently been demonstrated that myogenic tone is attenuated in the cerebral circulation of EC-specific Panx1 knockout animals, but is not altered in the mesenteric circulation of the same animals . The EC-Besides regulation of blood pressure, among other important aspects of the microcirculation, it is an important regulator of the acute inflammatory response. It was recently demonstrated that TNFα stimulation activates Panx1 in the venous, but not the arterial microcirculation . InteresAlso, in the microcirculation, there has been significant attention paid to the possible role of purinergic signaling from red blood cells (RBCs) to endothelium to induce vasodilation, especially during hypoxia. It had been hypothesized that Panx1 on RBCs was the mechanism by which ATP  could leave the RBC, bind to purinergic receptors on endothelium, and induce vasodilation. However, although Panx1 can be localized to RBCs , the rolApart from their role in inflammatory cell recruitment at the level of venules see , the fun−/−Apoe−/−Panx1 and −/−Apoe mice. The dye progressively spread throughout the lymphatic system to successive draining lymph nodes and finally the systemic circulation. The dye transport was considerably smaller in −/−Apoe−/−Panx1 mice than in control −/−Apoe mice. Moreover, tails of −/−Apoe−/−Panx1 mice showed increased diameters and increased interstitial fluid content than control −/−Apoe mice, suggesting that lymphatic flow is impaired in mice with ubiquitous deletion of Panx1. Finally, −/−Apoe−/−Panx1 mice had reduced dietary fat absorption with control animals. Collectively, these findings suggest a pivotal role for Panx1 in lymphatic function [The lymphatic system regulates tissue fluid homeostasis, trafficking of immune cells to draining lymph nodes and absorption of dietary fat. To investigate whether Panx1 affects lymphatic flow, drainage of interstitial fluids following injection of Evans Blue in the footpad was recently compared between function , and it Cx hemichannels and gap junctions have been studied widely in various cell types where sustained cell interactions occur. Some reports, however, indicate the presence of Cxs on the surface of some circulating cells, such as monocytes and T-cells, where gap junction and hemichannel functions control cellular functions ,153,154.2β1 physiological processes, including inflammation, atherogenesis, and cancer cell metastasis. Platelets adhere to collagens that are exposed at sites of arterial damage, initially via an indirect interaction with plasma von Willebrand factor (VWF), which through binding to the platelet glycoprotein (GP) Ib-V-IX receptor complex, a short-lived interaction that therefore serves to slow platelets, allows subsequent direct binding to platelet collagen receptors GPVI and integrin α2β1 A [155,15collagen , while c2β1), and causes aggregation through the binding of plasma fibrinogen to integrin αIIbβ3 [Collagen binding to GPVI causes receptor clustering and the tyrosine phosphorylation of the Fc receptor γ-chain ,159 by Sn αIIbβ3 ,163.2. Following the binding of fibrinogen to integrin αIIbβ3 and collagen to integrin α2β1, outside-in signaling through these integrins also contributes to sustained platelet activation and irreversible thrombus formation [A rapid and full platelet response is ensured through the autocrine and paracrine actions of factors that are released by activated platelets such as ADP and thromboxane Aormation ,165. Thr2, short-lived molecules that exert powerful inhibitory effects on platelets through the stimulation of cyclic nucleotide-dependent intracellular signaling [A reactive system such as this, which incorporates many positive feedback mechanisms, requires precise control to prevent un-needed platelet activation. The healthy endothelium produces molecular signals, NO and prostaglandin Iignaling .Inappropriate platelet activation, for example, at the site of atherosclerotic plaque formation or rupture, results in the exposure of platelets to activatory substances, including collagens, resulting in thrombosis and the occlusion of blood flow. As the principle cause of myocardial infarction and ischemic stroke, platelets represent an important therapeutic target .In the last 20 years, substantial progress has been made in understanding the molecular mechanisms that control platelets, and this is beginning to impact in the development of new therapeutic approaches. These advances have largely involved study of traditional intra-cellular signaling, the identification of key activatory or inhibitory signals, cell surface receptors required to respond to these signals and the intracellular signaling pathways or networks that these control. While platelets are singular circulating cells, activation and thrombus formation bring them into close proximity for prolonged periods, and increasing evidence supports the importance of sustained inter-platelet communications within the thrombus derived largely through integrin outside-in signaling, with additional contributions from, for example, Eph family kinases and ephrin counter-ligands ,168,169.Messenger RNA profiling of megakaryocytes, the precursor bone marrow cells from which platelets form, revealed that these cells likely possess notable levels of Cx37, with additional expression of Cx40 and Cx62. Indeed, Cx37 mRNA and protein were first reported to be expressed in human and mouse platelets, and were found to be present at the cell surface ,173. Sca37,43Gap27). Clot retraction responses were also inhibited in the presence of inhibitor, or the absence of Cx37 (using blood from Cx37-deficient mice) suggesting that gap junctional coupling mediates physiological responses within platelets.The transport of dye between platelets was first demonstrated following micro-injection of neurobiotin, and transfer into surrounding platelets . Gap junThere currently exists a difference of experimental conclusions drawn from the study of Angelillo-Scherrer, who reported modestly elevated platelet aggregation responses on Cx37-deficient mouse platelets and following the use of inhibitory peptides with human platelets , while V37,43Gap27 [40Gap27 (unpublished observation) into mice prior analysis of laser induced thrombosis in cremaster muscle arterioles was found to result in diminished thrombotic responses. Diminished thrombus formation appears to be at the expense of haemostatic control, since bleeding times were extended modestly. It is interesting to note the Cx37-deficient mice were found to exhibit reduced survival time using a thrombo-embolism model, induced by intravenous injection of collagen and adrenalin [The use of flow cytometry gated to examine the function of individual platelets revealed that platelet activation, prior to platelet-platelet contact is inhibited in the absence of functional Cx37, which is suggestive of important roles for Cx hemichannels in the initiation of platelet responses ,174. Whe,43Gap27  or 40Gapdrenalin , which mRecent analysis of the megakaryocyte (and therefore the platelet) channelome revealed that among a range of cell surface channel proteins, Panx1 is also likely to be expressed. A series of pharmacological approaches using selective mimetic peptide inhibitor , subsequCx gene possessed by volunteers, with the CC genotype associated with modestly increased platelet function [It is fair to ask, are the effects of platelet Cxs likely to be physiologically important, or do they contribute to cardiovascular disease risk? The clearest indication that gap junction and hemichannel function may be important stem from studies that have explored the effects of common gene SNPs in the human population. As discussed before , a SNP ifunction . These dThree variants exist in the human Panx1 coding sequence. These result in a Q5H variant at the N-terminus, a I272V variant within the 4th transmembrane domain and deletion of amino acids 401 to 404 at the CT due a splice variant. In a population of 96 male Caucasian volunteers the splice variant was not detected . Two thiThe numbers of subjects included in these studies were relatively small, and more detailed analysis of the relationship between platelet Cx and Panx polymorphisms and variability in platelet responsiveness would be required to confirm these observations. Current data are, however, consistent with a role of Cxs and Panxs in the regulation of haemostasis and potentially thrombosis, allowing the potential development of new strategies for the prevention of thrombosis, or other conditions in which these cells are implicated.It is now well established that Cxs have an important function in the control of blood flow distribution and tissue homeostasis, as well as in pathologies that involve a tight regulation and coordination between cells in the blood vessel wall and circulating blood cells such as atherosclerosis and hypertension. Strong evidence now also supports an important role for gap junctions and Cx hemichannels in the control of haemostasis and thrombosis, although many questions remain to be addressed in this field. More insight into the nature of molecular signals that are transported through gap junctions and hemichannels will be required in order to tease apart the basis of their ability to modulate diverse aspects of vascular function. Systems biology approaches have revealed exquisite detail regarding the architecture of platelet thrombi, which are organized into a densely packed core, surrounded by a less densely packed shell, which is sensitive to the actions of anti-platelet drugs . It is p2+ are generally assumed to be the most relevant signaling mechanisms mediated by both Cx hemichannels and Panx1 channels in vascular (patho)physiology. While recent years have shown great progress in the knowledge on Cx43 protein domains involved in gap junction channel vs. hemichannel gating [The release of ATP or Cal gating , one of l gating . Perhaps


In addition, their action can occur through interaction with other TFs and the cellular transcription machinery. The current genome sequences available reveal a relatively large number of WRKY genes, reaching hundreds of copies. Recently, functional genomics studies in model plants have enabled the identification of function and mechanism of action of several WRKY TFs in plants. This review addresses the more recent studies in plants regarding the function of WRKY TFs in both model and crop plants for coping with environmental challenges, including a wide variety of abiotic and biotic stresses.WRKY transcription factors (TFs) are responsible for the regulation of genes responsive to many plant growth and developmental cues, as well as to biotic and abiotic stresses. The modulation of gene expression by WRKY proteins primarily occurs by DNA binding at specific  Plants are continuously exposed to environmental stresses, such as variations in temperature, nutrient content and availability in the soil, rain availability, and pest/pathogen attacks. Their exposure to this range of stresses unleashes responses at various levels. At the molecular level it activates signaling cascades responsible for inducing or repressing target gene expression. Transcription factors (TFs) are proteins that regulate gene transcription, and any change in their activity dynamically alters the transcriptome, causing metabolic and phenotypic changes in response to a given environmental stimulus .cis-regulatory elements (CREs), which are short sequences present in the promoter regions of genes. TFs interact with CREs, other TFs, and with the basal transcription machinery in order to regulate the expression of target genes , protein kinase cascades that are phosphorylated in sequence and activate a series of proteins, and ion channels that change ionic features of cells .An increasing number of recent studies have indicated that WRKY proteins are also involved in a variety of other plant specific reactions such as senescence   based on additional structural motifs conserved outside the WRKY domain. The proteins from Group III show a WRKY domain containing different zinc-finger motifs Cys2-His/Cys Cys2-His2 (WRKY proteins share a DNA binding domain of about 60 amino acids that contains an invariable sequence WRKYGQK (from which the domain was named - WRKY) and a zinc-finger domain-like  and in Metazoa (Dictyostelium discoideum), suggesting an origin of WRKYs predating the origin of plants  modulate many plant signaling responses, including response to stresses, making a connection between stimuli perception and molecular cell responses  mRNA was detected, which was also observed in a mpk4-wrky33 double mutant. This suggested that AtWRKY33 acts with MPK4 in nuclear complexes that depend on MKS1, an MPK4 substrate. When infected with Pseudomonas syringae, MPK4 is activated and phosphorylates MKS1, which binds to AtWRKY33. That complex binds to a PAD3 promoter, which itself encodes an enzyme for camalexin synthesis  led to in vivo AtWRKY46 responses. When overexpressed, it raised basal plant defense, as reflected by the increase in promoter activity of NHL10 (a PAMP-responsive gene), in a MAPK-dependent pathway. A mechanism of plant defense controlled by AtWRKY46 in a MAPK-mediated pathway was suggested  sites that can be putatively phosphorylated by MAP kinases  plays a variety of roles in plant development, bud and seed dormancy, germination, cell division and movement, leaf senescence and abscission, and cellular response to environmental signals . During Arabidopsis, WRKY proteins act as transcription activators or repressors. Through single, double, and triple mutants, as well as lines overexpressing WRKY genes analyses, it was shown that AtWRKY18 and AtWRKY60 have positive effects on the ABA sensitivity of plants regarding inhibition of seed germination and root growth and increase in the sensitivity to salt and osmotic stress. AtWRKY40 antagonizes with AtWRKY18 and AtWRKY60 concerning the effect caused on the sensitivity of plants to ABA and abiotic stresses, and both genes are rapidly induced by ABA, while induction of AtWRKY60 is delayed by ABA (REGULATORY COMPONENT OF ABA RECEPTOR (RCAR), ABA INSENSITIVE 1 (ABI1) and ABI2, together with ABSCISIC ACID RESPONSIVE ELEMENTS-BINDING FACTOR (ABF) were found differentially expressed in the Atwrky40 mutant indicating that they are directly regulated by AtWRKY40  gene, which encodes for a phosphate carrier protein from root to shoot  were developed. AtbHLH17 (AtAIB) and AtWRKY28 are TFs known to be up-regulated under drought and oxidative stress, respectively. The transgenic lines exhibited enhanced tolerance to NaCl, mannitol, and oxidative stress. Under mannitol stress condition, significantly higher root growth was observed in transgenic plants. Growth under stress and recovery growth was substantially higher in transgenic plants exposed to gradual longer desiccation stress conditions. The TFs AtNAC102 and AtICE1, which contain either bHLH or WRKY binding cis-elements, showed enhanced expression in transgenic plants under stress. However, genes lacking either one of the two motifs did not differ in their expression levels in stress conditions when compared to wild type plants  enhanced the tolerance to salt stress by improving the plants’ ability to selectively uptake Na+ and K+ and maintain low Na+/K+ in leaves and roots of transgenic plants  also demonstrated salinity tolerance. Transgenic lines were able to develop roots, increase fresh weigh under salt stress, as well as showing an increase in antioxidative enzymes activity, and lower Na+ content and Na+/K+ ratio compared to wild type plants. Also, an enhanced expression of RtWRKY1 was detected in plants treated with ABA . In addition, GhWRKY27a expression was increased by ABA treatment , fusion of the gene promoter induced by heat shock Arabidopsis for AtWRKY39 gene, which positively regulates the signaling pathways activated by salicylic (SA) and jasmonic (JA) acids and mediates responses to heat stress  and AtMBF1c (related to SA) genes showed lower regulation. On the other hand, the overexpression of AtWRKY39 increased the expression of these genes.Heat stress was also studied in the Arabidopsis transgenic lines overexpressing CsWRKY46, a cucumber WRKY, showed higher survival rates of freezing seedlings at 4 °C. Hence, tolerance is suggested to be an ABA-dependent process, since an effect of exogenous ABA in transgenic lines was observed , with severe symptoms when infected  and drought stresses . The defense mechanisms are controlled by signaling molecules, such as salicylic acid (SA), jasmonic acid (JA), and ethylene, or by combinations of these signaling compounds  and AtPBS3 (AVRPPHB SUSCEPTIBLE 3) that plays an important role in SA metabolism (ICS1 promoter::β-glucuronidase (GUS) genes in protoplasts co-transfected with 35S::WRKY28 showed that overexpression of AtWRKY28 resulted in a strong increase in GUS expression. Moreover, RT-qPCR analyses indicated that the endogenous AtICS1 and AtPBS3 genes were highly expressed in protoplasts overexpressing AtWRKY28 or AtWRKY46, respectively. Electrophoretic mobility shift assays identified potential WRKY28 binding sites in the AtICS1 promoter positioned at 445 and 460 base pairs upstream of the transcription start site. Mutation of these sites in protoplast transactivation assays showed that these binding sites are functionally important for activation of the AtICS1 promoter. Chromatin immunoprecipitation assays with hemagglutinin-epitope-tagged WRKY28 showed that the region of the AtICS1 promoter containing the binding sites at 445 and 460 bp was highly enriched in the immunoprecipitated DNA , resistance to pathogen was found  and AtJAZ5, which encodes two important repressors of the JA signaling pathway by binding directly to their promoters  and providing increased resistance to the pathogenic bacteria Pseudomonas syringae . Studies on overexpression of WRKY genes furthermore indicated that these lines, besides displaying a particular pathogen resistance, also showed tolerance to abiotic stresses (Table S3).The Arabidopsis, the constitutive overexpression of the OsWRKY45 transgene confers a number of properties to transgenic plants. These properties include significantly increased expression of PR genes, enhanced resistance to the bacterial pathogen Pseudomonas syringae tomato DC3000, enhanced tolerance to salt and drought stresses, decreased sensitivity toward ABA signaling during seed germination and post-germination processes, and modulation of ABA/stress-regulated genes during drought induction. In addition, higher levels of OsWRKY45 expression in transgenic plants correlate positively with the strength of the abiotic and biotic responses mentioned above. More specifically, the decreased ABA sensitivities, enhanced disease resistance, and drought tolerances may be attributed, in part, to stomata closure and induction of stress-related genes during drought induction  showed enhanced sensitivity to Botrytis but enhanced resistance to Pseudomonas syringae pv tomato strain DC3000 through the regulation of SA-related gene expression (Nicotiana benthamiana overexpressing GhWRKY27a, while exhibiting reduced resistance to Rhizoctonia solani infection (Transgenic WRKY genes indicate tolerance to abiotic stresses, resistance to biotic stresses, or both. On the other hand, in studies where WRKY genes have been silenced, a sensitivity reaction to abiotic stresses and susceptibility to biotic stresses was observed. Some rare cases of negative regulation concerning stress were also reported.Most studies regarding the response of transgenic plants overexpressing The accurate regulation and fine adjustment of WRKY proteins during plant responses to stress contribute to the establishment of complex signaling networks, and the important roles of WRKY proteins in these networks make them potential candidates for conferring tolerance  in respoIn addition, considering the large number of genes of the WRKY TF family present in the genome of plant species, WRKY functions and interactions among the family members and WRKY protein kinases in the cascade of signaling need to be clarified.The development of advanced techniques of molecular biology, such as the combined use of microarrays, immunoprecipitation assays and sequencing techniques, may on a large scale, determine the DNA WRKY protein binding sites and their interaction with other proteins in signaling pathways. In addition, the function of WRKY in plants in response to environmental challenges might be clarified.


Misfolding protein diseases are a wide class of disorders in which the aberrantly folded protein aggregates accumulate in affected cells. In the brain and in the skeletal muscle, misfolded protein accumulation induces a variety of cell dysfunctions that frequently lead to cell death. In motoneuron diseases (MNDs), misfolded proteins accumulate primarily in motoneurons, glial cells and/or skeletal muscle cells, altering motor function. The deleterious effects of misfolded proteins can be counteracted by the activity of the protein quality control (PQC) system, composed of chaperone proteins and degradative systems. Here, we focus on a PQC system component: heat shock protein family B  member 8 (HSPB8), a chaperone induced by harmful stressful events, including proteotoxicity. In motoneuron and muscle cells, misfolded proteins activate HSPB8 transcription and enhance HSPB8 levels, which contributes to prevent aggregate formation and their harmful effects. HSPB8 acts not only as a chaperone, but also facilitates the autophagy process, to enable the efficient clearance of the misfolded proteins. HSPB8 acts as a dimer bound to the HSP70 co-chaperone BAG3, a scaffold protein that is also capable of binding to HSP70 (associated with the E3-ligase CHIP) and dynein. When this complex is formed, it is transported by dynein to the microtubule organization center (MTOC), where aggresomes are formed. Here, misfolded proteins are engulfed into nascent autophagosomes to be degraded via the chaperone-assisted selective autophagy (CASA). When CASA is insufficient or impaired, HSP70 and CHIP associate with an alternative co-chaperone, BAG1, which routes misfolded proteins to the proteasome for degradation. The finely tuned equilibrium between proteasome and CASA activity is thought to be crucial for maintaining the functional cell homeostasis during proteotoxic stresses, which in turn is essential for cell survival. This fine equilibrium seems to be altered in MNDs, like Amyotrophic lateral sclerosis (ALS) and spinal and bulbar muscular atrophy (SBMA), contributing to the onset and the progression of disease. Here, we will review how misfolded proteins may affect the PQC system and how the proper activity of this system can be restored by boosting or regulating HSPB8 activity, with the aim to ameliorate disease progression in these two fatal MNDs. Proteotoxic stress associated with aberrantly folded (misfolded) protein production is one of the factors thought to be deeply involved in the pathogenesis of several neurodegenerative diseases (NDs), including motoneuron diseases (MNDs). Amyotrophic lateral sclerosis (ALS) and spinal and bulbar muscular atrophy (SBMA) are two different types of MNDs clearly linked to the aberrant folding behavior of proteins in their conformationally unstable wild type (wt) or mutated forms . These aThe PQC system comprises a large number of factors, which may act specifically in some subcellular compartments  or that are expressed in a cell and tissue specific manner. The chaperone pathways alone comprise more than 180 different chaperones and their co-regulators, while the two major degradative pathways involved in the PQC system comprise more than 600 components (in the case of the ubiquitin-proteasome system) and at least 30 different components (in the case of the autophagy system) . The chaAs mentioned in the introduction, ALS and SBMA are typically considered to be protein misfolding diseases, in which unstable proteins or their mutated forms tend to aggregate impairing motoneuronal or muscle functions.From a clinical point of view, the two diseases present some difference, especially in the type of motoneurons affected. ALS is characterized by the loss of both upper and lower motoneurons and the regions affected are the brain motor cortex, the brainstem and anterior horns spinal cord motoneurons. In some fALS, alteration in the fronto-temporal regions are present and correlates with a mixed phenotype involving motor dysfunction and frontolateral temporal dementia (FLTD) . In ALS,ALS mainly appears as sporadic forms (sALS), but about 10% of the cases are inherited (fALS) and several mutated genes have already been described . Of noteAt present, it remains unclear if protein misfolding is the primary toxic event in ALS, or if it reflects alteration of specific intracellular pathways . Several recent data underline that autophagy dysfunction is implicated in ALS  and SBMAWith regards to SBMA, accumulation of autophagosome and reduced autophagic flux have been observed in cell and animal models of SBMA . InteresSeveral studies have proposed that HSPB8 is implicated both in ALS and SBMA, possibly by acting as a protective agent against disease onset and/or progression . It has Drosophila melanogaster models , the overexpression of the HSPB8 functional ortholog (HSP67Bc) protects from misfolded protein toxicity, while its downregulation has the opposite effect   and the abnormally translated dipeptides (DPRs) produced from the expanded GGGGCC repeated sequence of the or FLTD) .Very recently, it has been shown that HSPB8 is also able to maintain a correct dynamic behavior of stress granules (SGs), membraneless ribonucleoprotein (RNP) complexes that form via liquid-liquid phase separation (LLPS) .in vitro that are unstable and can mature with time into amyloid-like aggregates , etc.] . Similargregates . The ALSproteins . These oproteins , typicalproteins . Thus, Sproteins .So far, two mechanisms that decrease SG dynamics have been identified: (1) the presence of unstable aggregate-prone RBPs  and (2) The formation of cytoplasmic aggregates containing phosphorylated TDP-43 may arise also with SG-independent mechanisms, and can be induced by fibrillar fragments of aggregated TDP-43 itself or FUS . These aFrom the molecular point of view, the mechanism by which HSPB8 blocks the accumulation of misfolded proteins in cells affected in these MNDs is intriguing. As mentioned above, HSPB8 is a crucial component of the CASA complex. The intracellular levels of HSPB8 appear not to be sufficient to handle the large amounts of misfolded proteins that accumulate under certain stress conditions. In fact, the single overexpression of HSPB8 is able to restore a sufficient clearance of misfolded proteins, preventing their aggregation in cells. It is thus not surprising that the main activity of HSPB8 is to act as an autophagy facilitator. The first proof for this action comes from the observation that HSPB8 strongly interacts with the Ile-Pro-Val (IPV) domains of BAG3 forming a stable complex , 2015. MIn vitro experiments also support this hypothesis, since both αB-crystallin and HSPB1 overexpression are able to suppress SOD1 aggregation  dynein has been detected in SOD1 aggregates and (ii) alteration of retrograde transport is present in transgenic SOD1-G93A mice even if the legs at odd angles (Loa) mutation in cytopasmic dynein could be protective in transgenic SOD1-G93A mice where it delays disease onset and extends the life span . MoreoveDNAJB6 has been identified in human diseases linked to aberrant protein aggregation, like Limb-girdle muscular dystrophies (LGMDs)  and engulfed by the lipidated LC3-II into nascent autophagosome for clearance [see ]. This m (LGMDs) . Of note (LGMDs) , leadingde novo synthesis of HSPB8 and BAG3  in a HSPB8-dependent (even if not exclusive) manner . Since cOther small molecules that are able to induce HSPB8 expression have been characterized. One is trehalose, a non-toxic natural compound well-known for its ability to induce autophagy , whose mEstrogens and selective estrogen receptor modulators (SERMs) are also potent activators of HSPB8 expression , and thiGeranylgeranylacetone (GGA), also known as teprenone, which is an inducer of several HSPs, is also a potent upregulator of HSPB8 expression, and has been proven to be able to decrease the formation of amyloid oligomer and aggregates in desmin-related cardiomyopathy .Finally, one of the best known regulators of HSPB8 (and BAG3) is the NF-κB transcription factor, which is generally activated in the recovery period that follows a heat shock . Even ifIn conclusion, biochemical and immunohistochemical data obtained by using cell and animal (mouse and Drosophila) models of ALS and other neurodegenerative diseases support the interpretation that HSPB8 has a prominent role in counteracting the toxicity of misfolded proteins in MNDs and may be fundamental in the maintenance of the delicate equilibrium that regulates the routing of proteins to autophagy and to the proteasome. This is further suggested by the finding that HSPB8 and BAG3 are upregulated in the postmortem tissues from patients affected by several types of protein conformational diseases, specifically in the regions interested by neurodegeneration . HSPB8 mPR, RC, MG, VC, and AP designed and wrote the manuscript, and critically discussed all the sections of this minireview. RC prepared the figure. MEC, VF, BT, EC, MC, EM, MP, and SC critically revised the manuscript and figure. All authors have provided final approval of the version to be published.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.


Iron and zinc are essential micronutrients required for growth and health. Deficiencies of these nutrients are highly prevalent among populations, but can be alleviated by supplementation and food fortification. Cross-sectional studies in humans showed positive association of serum zinc levels with hemoglobin and markers of iron status. Dietary restriction of zinc or intestinal specific conditional knock out of ZIP4 (SLC39A4), an intestinal zinc transporter, in experimental animals demonstrated iron deficiency anemia and tissue iron accumulation. Similarly, increased iron accumulation has been observed in cultured cells exposed to zinc deficient media. These results together suggest a potential role of zinc in modulating intestinal iron absorption and mobilization from tissues. Studies in intestinal cell culture models demonstrate that zinc induces iron uptake and transcellular transport via induction of divalent metal iron transporter-1 (DMT1) and ferroportin (FPN1) expression, respectively. It is interesting to note that intestinal cells are exposed to very high levels of zinc through pancreatic secretions, which is a major route of zinc excretion from the body. Therefore, zinc appears to be modulating the iron metabolism possibly via regulating the DMT1 and FPN1 levels. Herein we critically reviewed the available evidence to hypothesize novel mechanism of Zinc-DMT1/FPN1 axis in regulating intestinal iron absorption and tissue iron accumulation to facilitate future research aimed at understanding the yet elusive mechanisms of iron and zinc interactions. Iron and zinc are essential micronutrients required for growth and sustained health. Physiologically, iron is defined as type 1 nutrient, while zinc is a type 2 nutrient ,4. PhytiIron and zinc combined supplementation trials in humans and animal models have revealed negative interactions, but there are conflicting data on the direction and magnitude of these interactions ,7. It haHere, we have reviewed the mechanism of iron and zinc homeostasis and their interactions both at the level of intestinal absorption and tissue mobilization in the context of zinc status. From the available evidence, it appears that zinc-DMT1/FPN1 axis is a critical determinant of zinc-deficiency-induced changes in iron homeostasis and helps understand the mechanism of iron and zinc interactions.In mammals, iron is highly conserved and there are no obligatory pathways for its excretion. The basal losses of iron through shedding of intestinal cells, sweat, and urine, and increased demand due to infant and adolescent growth spurts and pregnancy is compensated by the concurrent modulation of intestinal iron absorption. In addition, iron is stored and recycled in the body and thus counters short-term dietary inadequacies . The mec3+ at the ferritin shell before being stored . Zi. Zi40]. The identification of two families of zinc transporters, namely ZIP (increases cytosolic zinc) and the ZnT proteins (decreases cytosolic zinc), has contributed considerably to the understanding of both intestinal and systemic zinc homeostasis ,46,47. ZEarly studies using radio-tracer methods identified that the exocrine pancreas plays a functional role in zinc excretion. Indeed, pancreas, intestine, and liver have been identified to have very high turnover rates of zinc . In addin = 1185) found low serum zinc levels among anemic subjects, and furthermore, serum zinc levels were significantly and positively correlated with hemoglobin [acrodermatitis enteropathica, a rare genetic disease characterized by zinc deficiency [Clinical studies in human subjects indicated that serum zinc levels correlate with iron status markers. Hemoglobin, plasma ferritin, mean corpuscular volume (MCV), and red cell distribution width (RDW) were found to be higher in zinc sufficient (>100 µg/dL) compared to zinc deficient (<100 µg/dL) subjects . Similarmoglobin . Other smoglobin ,13. In amoglobin . These sficiency , therefoStudies in experimental animals demonstrated development of iron deficiency anemia and tissue iron accumulation during zinc deficiency. For instance, studies in rats given low zinc diets led to a reduction in iron status parameters such as hemoglobin and RBC number, which could be either due to reduced erythropoiesis or increased catabolism ,60. SimiIt has also been hypothesized that the zinc might induce iron homeostasis via regulation of hepatic hepcidin expression ,64. HepcSurprisingly, there are no studies measuring iron absorption in relation to zinc status in humans, possibly due to lack of reliable biomarker of zinc deficiency. However, a study in suckling rat pups demonstrated that during early infancy (at day 10 of parturition) zinc supplementation increases the DMT1 and FPN1 mRNA and protein levels, but these effects are not observed in late infancy (at day 20 of parturition) . This stThe homeostatic mechanisms controlling iron and zinc metabolism compensate for fluctuations in dietary exposure. However, chronic consumption of foods that have low levels of these dietary metals or abundant concentrations of dietary inhibitors leads to negative balance or deficiency. Considering the high prevalence of anemia and zinc deficiency in populations, supplementing iron and zinc together could be an ideal strategy. Studies in fasting human subjects have indicated a dose-dependent decrease in zinc absorption (25 mg dose) as measured by area under the curves during 4 h time period post-dosing, when supplemented along with non-heme iron, at 2–3-fold molar excess, but not with heme iron . The inhIf the interactions of iron and zinc occur at a specific protein site, zinc would also be expected to inhibit the absorption of iron. Indeed, zinc at high doses does reduce the absorption of iron in adult human subjects when fed with liquid, but no such effects were seen when given in a meal . A revieA meta-analysis of longer-term zinc supplementation trials indicated no impact of zinc on hemoglobin . FurtherIt was thought initially that iron and zinc, due to their similar atomic radius and oxidation state, might compete for intestinal absorption at divalent metal ion transporter-1 (DMT1), a proton coupled apical iron transporter in intestinal cells . HoweverWe have demonstrated in Caco-2 cells that iron and zinc inhibit the absorption of each other ,19. HoweAs described above, unlike iron, whose homeostasis is regulated by controlling the absorption zinc homeostasis is exclusively regulated by controlling the excretion of endogenous zinc. Although zinc excreted via multiple routes such as urine, sweat, and semen, the enteric secretions constitute a major route of endogenous zinc secretion. Enteric zinc is formed of a combination of dietary zinc and zinc from endogenous secretions derived from bile and pancreatic juice ,43. In rIt is estimated that 2 mg of zinc is lost through pancreatic secretions per day which corresponds to ~12 µmol/L zinc considering the 2.5 L juice produced per day ,85. In aIt is clear from the above observations that zinc status has a marked impact on iron absorption and metabolism. It is possible that this has physiological relevance and metabolic advantage. It is known that iron and zinc compete with various metabolic proteins due to their similar physico-chemical properties. It is established that iron is a pro-oxidant while zinc is seen as an antioxidant . TherefoThe available evidence from experimental animals, cross-sectional studies in human subjects, and genetic studies clearly point to an association of whole-body zinc status with iron homeostasis. Particularly, underlying zinc deficiency appears to induce iron deficiency by mechanisms that block either intestinal absorption or mobilization of iron from tissues. The in vitro studies in intestinal cell culture models and studies in animals point to a role for zinc in modulating DMT1 and FPN1 expression, respectively. It is therefore possible that compromised zinc status leads to a reduction in pancreatic zinc content, which in turn reduces intestinal iron absorption via a decrease in DMT1 and FPN1 expression. Thus, the net effect of zinc deficiency is mechanistically linked with development of iron deficiency, induced by both reduced intestinal iron absorption and decreased mobilization of iron from storage sites.Although this review highlights the cross-talk between zinc with intestinal absorption and tissue mobilization of iron, further investigations are warranted to understand the nexus between these important minerals. For example, there is no direct demonstration of the impact of underlying zinc status  on iron absorption or hepcidin levels in humans. A typical stable isotope-based iron bioavailability study, with short term dietary restriction of zinc, could be a way forward in this direction. In addition, studies are needed to understand the regulation of hepatic iron metabolism by zinc, particularly those regarding the expression/activity of matriptase-2, BMP signaling, and ferritinophagy may shed further light on the impact of zinc on systemic iron metabolism. Since PI3K activation is required for induction of iron absorption (at least in in vitro studies), and dietary ligands such as phytic acid and polyphenols are known inhibitors of this pathway, further studies are needed to understand if the contribution of these inhibitors to the regulation of iron absorption extends beyond chelation of these metals. In addition, studies are also needed to understand both short- and long-term consequences of dietary zinc restriction on iron metabolism, particularly the mRNA and protein expression of iron metabolic proteins and the effects on distribution and concentrations of metals between different tissues.It is generally accepted that iron and zinc deficiencies coexist, and it is clear from the above evidence that they do interact negatively when supplemented together. Since baseline zinc deficiency also adversely affects the iron absorption, correction of zinc status might improve the outcomes of iron therapy in populations at risk of zinc deficiency. Furthermore, since zinc rapidly induces the intestinal iron absorption (4 h), alternate supplementation of zinc followed by iron with a time gap is also expected to improve the response to iron therapy, and indeed such evidence already exists .