Psoriasis is a chronic, painful, disfiguring and non-contagious skin disease that has globally affected at least 200 million patients. In general, mild to moderate psoriasis patients will be treated by chemical drugs or Chinese medicine, while targeting systemic biological drugs have been successfully developed with good efficacy but high cost burden to patients with severe psoriasis. Since the underlying mechanisms of psoriasis are not well understood, in this review, psoriasis pathogenesis and clinical therapeutic principles by modern medicine and Chinese medicine are extensively described. Based on the data from the China Food and Drug Administration, the majority of chemical drugs are utilized as the topical formulations, while Chinese medicines are mainly delivered by an oral route, suggesting that the market for topical preparations of Chinese medicine to treat psoriasis is worth to exploration. Moreover, considering the unique clinical therapeutic theory and successful clinical application of Chinese medicine in the treatment of psoriasis, we believe that development of new small molecule drugs based on Chinese medicine will be a promising strategy to reduce therapeutic costs and improve safety for psoriatic patients. Psoriasis is a chronic, painful, disfiguring and non-infectious skin disease that is believed to be an autoimmune inflammation disorder with unclear underlying mechanism . PsoriasPsoriasis could be divided into five types . PsoriasTwenty-five years ago, people thought that, psoriasis was caused of excessive proliferation of skin cells and the chronic inflammation was just the side effect. In 1994, Krueger, a cell biologist at the Rockefeller University, started working on the origins of psoriasis. His team administered a compound that could target the immune-related cells without affecting the normal skin function of patients. The results showed a great improvement of the skin condition, and some individual cases with mild trauma were even cured. This experiment proved that psoriasis was mainly caused by immune system cells and was not the side effect of cell proliferation. The research of Krueger involved first separating the different roles of keratinocytes and T cells .The main characteristics of psoriasis are excessive proliferation of keratinocytes and being easy to relapse. It is reported that this disease might involve many tissues in the muscles, joints, alimentary tract and even eyes . The carIL-17 is a proinflammatory cytokine produced by activated T cells, leading to the generation of inflammation . IL-23 b+ dendritic cells exist at the skin lesions of psoriasis. The mRNAs expression levels of subunits of IL-23 are also relatively higher  and Dichlorodiethyl sulfide, and they found comparatively equal efficacy. For oral prescriptions, the clinical efficacy of HuoXueSanYu Decoction  were found to be comparable to acitretin , for the treatment of psoriasis vulgaris. JiaWeiHuangLianJieDu Decoction  was reported to alleviate the symptoms in 45 of 50 psoriatic subjects.Various remedies used to treat psoriasis were recorded in Chinese medicinal classics, like 75 cases  made a cecoction   performed better efficacy and a lower relapse rate than combined with an oral placebo. ZaoShiKuShen Decoction  was jointly applied with 0.03% Tacrolimus, which showed 14% higher efficacy than that of 0.03% tacrolimus alone in a 100-patient clinical trial.In most cases, the formula for anti-psoriatic treatment consists of different kinds of herbs, which are used as adjuvant therapy or joint with other drug as combined treatment. Anti-psoriatic herb compound prescriptions commonly combine with Narrow Bound Ultra Violet B(NB-UVB) light to achieve higher efficacy. A study  analyzed A study  demonstrecoction   exhibited a successful treatment on pediatric psoriasis [Rhizoma Zingiberis (Ganjiang), Radix Salviae Miltiorrhizae (Danshen), Radix Astragali (Huangqi), Ramulus Cinnamomi (Guizhi), Radix Paeconiae Alba (Baishao), Radix Codonopsis Pilosula (Dangshen) and Semen Coicis (Yiyiren)) were reported to have better efficacy on plaque psoriasis [Some of the traditional prescriptions or active ingredients extracted from Chinese herbs have been developed into hospital agents with good efficacy. For instance, an soriasis . New Pulsoriasis . Herose soriasis .Radix Sophorae Flavescentis (Kushen) (Approved Number: Z14021231), another involving Fructus Psoraleae (Buguzhi) (Approved Number: Z41022361) and six topical agents. However, from 2004 to June 2012, the National Adverse Drug Reaction Monitoring Center has revealed 344 cases that appeared to have adverse reactions in that digestive system, skin and its affiliated glands, and the nervous system after being treated with systemic administered prescriptions, and 23 severe cases involved medicinal liver damage and gastrointestinal bleeding. Compared to systemic preparations, those for topical use showed less adverse events including hotness, redness, dryness or itching on the administered skin, which are usually reversible or self-healing.In addition, drugs approved by CFDA have also been studied, which contained herbal prescriptions and active composites extracted from herbs in various dosage forms, such as tablets, pills, creams, ointments, patches and injections. In the 34 approved drugs, the majority of them were orally administered two or three times per day, while 17 of them were the same extraction prepared in different dosage forms, such as formula Fufangqingdai, which has been prepared into tablets (Approved Number: Z20150034), pills (Approved Number: Z61020964), condensed pills (Approved Number: Z20080269) and capsules (Approved Number: Z20010157). There were also two injections, one containing Curcuma longa L., has been developed into various kinds of formulations due to its inhibition of several inflammatory enzymes. Studies have found that it exerted anti-psoriatic action probably via suppressing the expression of VEGF and iNOS [With huge potential for treating psoriasis, novel drugs involving pure ingredients extracted from Chinese herbs also have been explored. Curcumin, which is an active constituent in and iNOS . Additioand iNOS . Curcumiand iNOS . Fufangeand iNOS  and ethaand iNOS  were botand iNOS . Our preand iNOS .Indigo naturalis (Qingdai), significantly mitigated psoriatic lesions induced by imiquimod in mice. Meanwhile, it could suppress infiltration of inflammatory cells, proliferation of epidermal cells and expression of pre-inflammatory factors. In addition, in vitro studies demonstrated the inhibition effect on the expression of IL-17A and phosphorylation of STAT3 and JAK3, indicating that the expression of IL-17A from γδ T cells and immuno-inflammatory reactions can be inhibited by indirubin probably via mediating the JAK3/STAT3 signaling pathway [Paeonia lactiflora Pall. (Shaoyao), whose active compound is paeoniflorin, can significantly alleviate imiquimod-induced psoriatic lesions and inhibit the infiltration of inflammatory cells as well as the proliferation and differentiation of epidermal cells. Furthermore, it mainly targeted the regulation of Th cell factor expression, and inhibited the excretion of relevant cytokines probably via modulating the phosphorylation of STAT3 [rhizoma Smilacis Glabrae (Tufuling), showed suppressive effects on the differentiation of Th 17 cells and γδ T cells. Therefore, the content of IL-17 in lesions and peripheral blood was mediated. However, the specific underlying molecular mechanics remains unclear [Tripterygium wilfordii Hook. (Leigongteng) is a traditional medicinal herb that has been used for several centuries in China. Multi-glycoside from this herb could effectively disturb the formation of skin lesions in imiquimod-induced mice and inhibit the proliferation of epithelial cells and infiltration of T cells in lesion sites via downmodulating Th 17 function [Arnebiae Radix (Zicao), a blood-regulating herb, and its ingredient β, β-dimethyl acryloyl alkannin has demonstrated suppressing psoriasis-activated dendritic cells and inhibiting mRNA expression of inflammatory factors due to curbing the TLR7/8 pathway [Moreover, we have performed extensive studies to understand the underlying mechanisms of single constituents isolated from effective Chinese medicines on psoriasis treatment. For example, our recent studies revealed that indirubin, the active component of  pathway . Paeoniaof STAT3 . Astilbi unclear . Tripterfunction . Arnebia pathway , 64.Psoriasis not only affects people with itching and pain but also causes serious distress in their daily lives. Because of the abnormal appearance and texture of the skin, it is likely that psoriasis patients will be less likely to take part in social activities because of depression, so apparent skin treatment is more important. According to the CFDA data, the chemical formulations of psoriasis treatments are mainly topical drugs, while the traditional Chinese medicine formulations are mainly oral systemic drugs, suggesting that the market of topical preparations of Chinese medicine for the treatment of psoriasis is still waiting for exploration. At present, the most effective drug is biologics, but their cost, relative to chemical drugs or traditional Chinese medicine, is too high to be used for long term treatment. Looking for new technologies to reduce production costs is definitely a development trend for monoclonal antibody drugs. On the other hand, compared to chemical and biological treatments, traditional Chinese medicine and their active extracts are much cheaper with fewer adverse reactions, which is more suitable for the treatment of less severe psoriasis. Therefore, the topical use of Chinese medicine or their small-molecule extractions for the treatment of psoriasis is a potentially promising new area for future exploration.


Eosinophils are multifunctional granulocytes that contribute to initiation and modulation of inflammation. Their role in asthma and parasitic infections has long been recognized. Growing evidence now reveals a role for eosinophils in autoimmune diseases. In this review, we summarize the function of eosinophils in inflammatory bowel diseases, neuromyelitis optica, bullous pemphigoid, autoimmune myocarditis, primary biliary cirrhosis, eosinophilic granulomatosis with polyangiitis, and other autoimmune diseases. Clinical studies, eosinophil-targeted therapies, and experimental models have contributed to our understanding of the regulation and function of eosinophils in these diseases. By examining the role of eosinophils in autoimmune diseases of different organs, we can identify common pathogenic mechanisms. These include degranulation of cytotoxic granule proteins, induction of antibody-dependent cell-mediated cytotoxicity, release of proteases degrading extracellular matrix, immune modulation through cytokines, antigen presentation, and prothrombotic functions. The association of eosinophilic diseases with autoimmune diseases is also examined, showing a possible increase in autoimmune diseases in patients with eosinophilic esophagitis, hypereosinophilic syndrome, and non-allergic asthma. Finally, we summarize key future research needs. The cells of the innate immune system can contribute to autoimmune diseases. Activation of innate immune cells by pathogen-associated molecular patterns and antigen presentation by dendritic cells can result in priming of autoreactive T and B cells and set off an adaptive immune response against self-antigens –3. PossiEosinophils are granulocytes that develop in the bone marrow in response to IL-5, with a minor role for IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-33 –15. IL-5The main chemotaxins for eosinophils are eotaxins, which homeostatically recruit eosinophils to the gastrointestinal tract, thymus, and uterus –21 and tA unique characteristic of eosinophils are their specific  granules. These are secretory vesicles with an electron-dense core and an electron-lucent matrix. Eosinophil granules contain four major granule proteins and numerous cytokines, chemokines, and growth factors  Figure . CytotoxEosinophils are extremely versatile effector cells that damage tissues or modulate the activity of other immune and stromal cells. One could envision many of these effector functions playing a role in the context of autoimmune diseases as well Box . Damage Box 1Damage of tissues by cytotoxic granule proteinsAntibody-dependent cellular cytotoxicityActivation of tissue remodeling and fibrosisAntigen presentationModulation of the adaptive immune responsePromotion of B cell responsesInduction of tissue repair processes.The ability of eosinophils to bind antibodies and consequently degranulate and kill cells links the adaptive autoimmune response to eosinophil effector functions. Eosinophils express complement receptors  and Fc rEosinophils also interact with stromal cells. Actively degranulating eosinophils are frequently found in areas of fibrogenesis, suggesting a potential profibrotic role –61. GranEosinophils can form extracellular DNA traps by quickly releasing mitochondrial DNA and granule proteins . These sin vitro experiments have demonstrated their ability to present antigen to and activate T cells  are easily identified by hematoxylin and eosin staining of histological sections due to the bright pink staining of the basic granules, which gave them the name eosinophils . Mouse bBullous pemphigoid is a blistering disease of the skin with a well-established autoimmune etiology . AutoantSeveral mechanisms by which eosinophils (and other granulocytes) contribute to lesion formation have been identified. Eosinophils in lesional skin were shown to degranulate, and granule proteins are deposited in blisters , 137. Thin vitro experiments, and animal models for a pathogenic role of eosinophils in bullous pemphigoid. In addition to the mouse model of passive antibody transfer, which reproduces blister formation but not eosinophil infiltration .Taken together, there is strong evidence from patient studies, ltration , a new mltration . Mice wiThe etiology of the inflammatory bowel diseases, Crohn’s disease and ulcerative colitis, is not fully understood. Evidence for the involvement of autoimmune processes exists for both , 149. BoHelicobacter hepaticus infection also reduced disease severity  or other mediators that affect the intestinal barrier. Thus, there is a strong evidence for a pathogenic role of eosinophils in inflammatory bowel diseases, particularly in ulcerative colitis.Eosinophilic granulomatosis with polyangiitis was first described by Churg and Strauss in 1951 . The disBlood and tissue eosinophilia are diagnostic criteria for EGPA, yet little is known about the pathogenic role of eosinophils in this disease . One reaBlood eosinophils in EGPA show an activated phenotype expressing high levels of CD69 and CD11b , 204. Moin vitro .The strongest evidence for a pathogenic role of eosinophils in EGPA comes from novel biological treatments that target IL-5 and thereby drastically reduce eosinophil levels. Two open-label trials with the anti-IL-5 antibody mepolizumab demonstrated its efficacy as a steroid-sparing agent and its ability to induce remission over 9 months –221. UpoSeveral key findings amount to moderate evidence for a pathogenic role of eosinophils in EGPA. (1) The number of eosinophils and serum ECP correlate with disease severity. (2) Eosinophil infiltration and degranulation in tissues causes organ damage. (3) A potential feed-forward loop between Th2 cells and eosinophils may propagate disease. (4) IL-5-targeted therapies showed beneficial effects.Myocarditis is the inflammation of the heart muscle with or without damage or necrosis of adjacent myocytes in the absence of an ischemic event . A wide Eosinophils form a major part of the inflammatory infiltrate in subtypes of myocarditis, namely in eosinophilic myocarditis and giant cell myocarditis. These subtypes are usually idiopathic. Eosinophilic myocarditis is associated with hypereosinophilic syndrome (HES) and EGPA, but it can also develop in the absence of eosinophilia. About one-third of EGPA patients and 20–50% of HES patients develop cardiovascular manifestations , 226–229−/−IL-17A−/−) results in severe eosinophilic myocarditis with about 50% fatality by day 21 , which is the major cause of heart failure in patients younger than 40 years and has a poor 5-year survival rate of less than 50% , 245. It−/−IL-17A−/− mice, cardiac expression of eotaxin-1 and -2 is highly increased compared to naïve mice or WT controls  is a demyelinating disease of the central nervous system (CNS) that usually affects the optic nerve and spinal cord. Lesions are necrotic, cavitary, and infiltrated with macrophages and granulocytes. NMO is an autoimmune disease. Anti-aquaporin 4 (AQP4) autoantibodies are present in the majority of patients. These pathogenic antibodies are highly specific for NMO and are one of the features that distinguish it from multiple sclerosis. NMO patients often carry multiple other autoantibodies, and there is a strong association with other autoimmune diseases. Moreover, NMO is much more common in women than men .Neuromyelitis optica has only recently been distinguished from multiple sclerosis with eosinophil infiltration being one of the distinctive features . The firin vitro experiments and a mouse model to determine the role of eosinophils in NMO  is the only approved drug for primary biliary cirrhosis patients. UDCA can delay disease progression and improve liver biochemistry . Of noteIn some cases, eosinophilia may precede the detection of liver pathology, suggesting that eosinophils are involved early in the disease processes. One study reports on four cases of asymptomatic women with eosinophilia detected during random investigation. All of them had elevated liver enzymes and were diagnosed with primary biliary cirrhosis . In anotSeveral rare diseases with a possible autoimmune etiology are associated with eosinophils. Usually only case reports or small case series are available for these diseases, making it very difficult to assign a pathologic or protective role to eosinophils.In vitro, IL-2 treatment of CD25+ eosinophils resulted in priming and increased release of ECP upon subsequent PAF stimulation. This suggests that eosinophils in patients with eosinophilic cellulitis may degranulate more easily. Indeed, extracellular MBP staining is readily observed in flame figures (Eosinophilic cellulitis (Wells’ syndrome) is a very rare skin disease characterized by recurrent edematous erythema. Eosinophilic cellulitis is potentially associated with EGPA, HES, UC, or other causes, but the etiology is unknown , 275. Th figures , 278 and figures . Eosinop figures . It is pEosinophilic fasciitis is characterized by thickening and inflammation of the fascia resulting in painful swelling and progressive induration of the skin and soft tissues , 282. ThThe fibroinflammatory IgG4-related disease can affect multiple organs including the pancreas, salivary and lacrimal glands, lungs, retroperitoneum, and other tissues , 289. WhEosinophils are a key feature of asthma, hypereosinophilic syndromes, and eosinophilic gastrointestinal diseases. An increased frequency of autoimmune diseases in patients with these eosinophil-associated diseases would be suggestive of a possible role for eosinophils in autoimmunity.vice versa. In a trial of mepolizumab therapy for HES, 5 of 78 patients under follow-up developed autoimmune diseases   . These dAutoimmune diseases may be associated with eosinophilic gastrointestinal diseases. A recent literature review summarized case reports of autoimmune connective tissue diseases  in patients with eosinophilic gastroenteritis . These pEosinophils are a prominent feature of allergic asthma and, to a lesser extent, non-allergic asthma . A potenTo date, there is no strong evidence for increased autoimmune diseases in patients with HES, asthma, or eosinophilic gastrointestinal disease, and only one study determined an increased risk in EoE patients. Future cross-sectional or cohort studies will be required to determine if the prevalence of autoimmune diseases is truly higher in patients with eosinophil-associated diseases.There is clear evidence for a pathogenic role of eosinophils in several autoimmune diseases Table . ProtectMultiple effector mechanisms have been identified Figure . DegranuParticularly for rare diseases, the evidence for eosinophil involvement is mostly based on case reports, which makes it difficult to exclude associations by chance. Because autoimmune disease patients may receive many drugs, it is worth considering that hypersensitivity reactions to drugs are often accompanied by eosinophilia. On the other hand, it is difficult to ascertain the role of eosinophils in patients treated with glucocorticoids, which are highly effective at reducing eosinophil numbers in blood and organs , 327. Eoin vitro or animal models has hampered research in several autoimmune diseases such as EGPA and primary biliary cirrhosis. In addition, epidemiological studies including larger patient cohorts will be required to determine whether autoimmune diseases are indeed increased in patients with eosinophil-associated diseases such as eosinophilic esophagitis, hypereosinophilic syndrome, or asthma. Finally, viewing and analyzing autoimmune diseases with eosinophil involvement as a group with possible shared mechanisms may advance our understanding and point to common processes.To verify some of the proposed mechanisms and potentially identify new mechanisms of eosinophil-mediated pathology or protection in autoimmune disease, animal models will aid greatly. The lack of ND reviewed the literature and wrote the manuscript. NR conceptualized the idea for the review and edited the manuscript. DČ edited the manuscript and contributed to defining the scope and content 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.


Obesity is a worldwide epidemic that predisposes individuals to metabolic complications, such as type 2 diabetes mellitus and non-alcoholic fatty liver disease, all of which are related to an imbalance between food intake and energy expenditure. Identification of the pathogenic molecular mechanisms and effective therapeutic approaches are urgently needed. A well-accepted paradigm is that crosstalk between organs/tissues contributes to diseases. Endothelial dysfunction characterizes metabolic disorders and the related vascular complications. Over the past two decades, overwhelming studies have focused on mechanisms that lead to endothelial dysfunction. New investigations, however, have begun to appreciate the opposite direction of the crosstalk: endothelial regulation of metabolism, although the underlying mechanisms remain to be elucidated. This review summarizes the evidence that supports the concept of endothelial regulation of obesity and the associated insulin resistance in fat, liver, and skeletal muscles, the classic targets of insulin. Outstanding questions and future research directions are highlighted. Identification of the mechanisms of vascular endothelial regulation of metabolism may offer strategies for prevention and treatment of obesity and the related metabolic complications. The triad of obesity, diabetes, and cardiovascular diseases (CVD) becomes a major public health threat because of the global shift toward an energy-rich and sedentary life style. The WHO predicts that overweight and obesity may soon become the most significant cause of poor health, in addition to malnutrition and infectious diseases . While hDespite decades of intense research, the major molecular pathogenesis behind complex metabolic diseases is unknown . A well-Endothelial cells form the lining of all blood and lymphatic vessels within the vascular network. The adult human body contains more than one trillion ECs, which weigh more than 100 g and cover a surface area of more than 1,000 square meters . As suchObesity contributes significantly to the development and progression of cardiovascular disease , which iObesity results from accumulation of white adipose tissue , 30. AdiResponding to caloric intake, adipose tissue expands. People who are prone to deposit visceral but not subcutaneous adipose tissue have a higher risk of metabolic disease . AlthougLinage tracing experiments demonstrated that the vascular endothelium of the adipose tissue gives rise to both white and brown fat cells , presumaIn vitro studies have shown that vascular-derived signals, e.g., endothelial-VEGF, directly affect the proliferation and differentiation of the surrounding pre-adipocytes, suggesting that angiogenesis can be a therapeutic target for obesity and metabolic diseases  are therapeutically potent progenitor cells because they possess properties of pericytes. ASCs in combination with EC establish functional multilayer vessels. Transforming growth factor (TGF)-β is secreted by EC  and is aEndocrine fibroblast growth factors (FGFs), such as FGF21, FGF15/19, and FGF23, are critical for maintaining whole-body homeostasis, because they operates in inter-organ endocrine signaling pathways that govern glucose, bile acid, and lipid metabolism , 55. FGFvia blocking unwanted endothelial activation -derived NO, the major endothelial-derived vasodilator, is central to endothelial regulation of insulin sensitivity in skeletal muscle, by stimulating blood vessel relaxation (in vascular smooth muscle cells) and perfusion  . In addiThere is evidence indicating that insulin delivery to skeletal muscle interstitium through the ECs is rate-limiting in insulin-stimulated glucose uptake in mice , 81 and Fatty acids represent a key energy source that is used by a number of tissues, which must be tightly controlled to avoid lipotoxicity induced by an excess of unoxidized fatty acids . ObesityThe liver is responsible for maintaining fasting glucose levels. Excessive accumulation of lipids in the liver impairs hepatic responsiveness to insulin, leading to elevated levels of glucose and insulin in the circulation, eventually chronic hyperinsulinemia . InsulinObesity and insulin resistance can cause NAFLD, which is characterized by liver metabolic dysregulation, such as hyperlipidemia . Recent l-amino acid-defined, and high-fat diet models that mimic human NAFLD indicated that LSEC injury functions as “gatekeeper” in the progression from simple steatosis to the early non-alcoholic steatohepatitis stage, and LSEC injury may activate Kupffer cells and hepatic stellate cells, which in turn results in chronic liver injuries While tissue- or organ-specific gene expression  has bee(2)Phenotypic heterogeneity is a central feature of the endothelium, referring to differences in the location of the conduit vessels  and their functions in serving specific organs  , 111. In(3)Endothelial cell-derived factors can be essential in maintaining organ homeostasis and regeneration. Could endothelium itself also operate as a rate-limiting apparatus  that re(4)in vivo? Or can specific parameters in this regard be identified by global assessment of gene expression profiles, epigenetic markers, and transcriptional factors?Severe impairment of endothelial function eventually promotes CVD. Non-invasive assessment of conduit vascular endothelial function is possible both in adults  and chil(5)Over the past two decades, many experimental or therapeutic drugs that improve CVD risk factors also improved endothelial functions . Do thesIn conclusion, the mechanisms underlying the endothelial-specific effects discussed here remain to be fully determined. Further translational studies to determine the clinical relevance of endothelial regulation of metabolism will provide greater insights that may ultimately lead to therapeutic advances against the increasing burden of the obesity pandemic and the associated metabolic disorders.JX contributed to the conception. JX, ML, and MQ wrote the article.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.


Neuroinflammatory response is primarily a protective mechanism in the brain. However, excessive and chronic inflammatory responses can lead to deleterious effects involving immune cells, brain cells and signaling molecules. Neuroinflammation induces and accelerates pathogenesis of Parkinson’s disease (PD), Alzheimer’s disease (AD) and Multiple sclerosis (MS). Neuroinflammatory pathways are indicated as novel therapeutic targets for these diseases. Mast cells are immune cells of hematopoietic origin that regulate inflammation and upon activation release many proinflammatory mediators in systemic and central nervous system (CNS) inflammatory conditions. In addition, inflammatory mediators released from activated glial cells induce neurodegeneration in the brain. Systemic inflammation-derived proinflammatory cytokines/chemokines and other factors cause a breach in the blood brain-barrier (BBB) thereby allowing for the entry of immune/inflammatory cells including mast cell progenitors, mast cells and proinflammatory cytokines and chemokines into the brain. These peripheral-derived factors and intrinsically generated cytokines/chemokines, α-synuclein, corticotropin-releasing hormone (CRH), substance P (SP), beta amyloid 1–42 (Aβ1–42) peptide and amyloid precursor proteins can activate glial cells, T-cells and mast cells in the brain can induce additional release of inflammatory and neurotoxic molecules contributing to chronic neuroinflammation and neuronal death. The glia maturation factor (GMF), a proinflammatory protein discovered in our laboratory released from glia, activates mast cells to release inflammatory cytokines and chemokines. Chronic increase in the proinflammatory mediators induces neurotoxic Aβ and plaque formation in AD brains and neurodegeneration in PD brains. Glial cells, mast cells and T-cells can reactivate each other in neuroinflammatory conditions in the brain and augment neuroinflammation. Further, inflammatory mediators from the brain can also enter into the peripheral system through defective BBB, recruit immune cells into the brain, and exacerbate neuroinflammation. We suggest that mast cell-associated inflammatory mediators from systemic inflammation and brain could augment neuroinflammation and neurodegeneration in the brain. This review article addresses the role of some atypical inflammatory mediators that are associated with mast cell inflammation and their activation of glial cells to induce neurodegeneration. Neuroinflammation is an inherent host-defense mechanism to protect and restore the normal structure and function of the brain against infection and injury  downregulates mir-7116-5p in microglia and potentiates TNF-α production and inflammatory responses leading to DA neuron damage  has been reported in AD patients indicating peripheral immune response in AD , corticotropin-releasing hormone (CRH), granulocyte macrophage-colony stimulating factor (GM-CSF), CCL2, dopamine, substance P (SP), histamine, tryptase, prostaglandins (PGD), leukotrienes, ROS, reactive nitrogen species (RNS) and nitric oxide (NO) selectively depending upon the tissue micro environment and the type of stimuli , SP, bradykinin, endorphin, endothelin, neurotensin, NO, vasoactive intestinal polypeptide (VIP) and urocortin  in an AD brain. Aβ1–42 is known to activate mast cells that could play an important role in AD pathogenesis , including increased NF-κB activation and greater cytokine production. Macrophages overexpressing UCP2 produce less ROS in response to LPS  phosphorylation, which is known to regulate prostaglandin synthesis  axis  formation and mast cell activation (Mori et al., Neurotransmitter dopamine is implicated in the pathogenesis of PD and in AD (Bisaglia et al., 2 into the brain (Ek et al., AD patients show abnormalities in peripheral tissues, immune and inflammatory cells in addition to the pathological changes observed in the brain regions (Khan and Alkon, + from glia or extracellular Aβ1–42 can activate mast cells to release neuroinflammatory mediators in PD and AD, respectively. Additionally, several inflammatory mediators from peripheral tissue can alter BBB, enter the brain and activate various neuroinflammatory pathways. Furthermore, inflammatory mediators released from activated microglia and astrocytes enter into peripheral system through defective BBB, activate, and recruit immune and inflammatory cells towards the inflammatory site in the brain. However, the influence of peripheral factors on neuroinflammation could vary from individual to individual and time to time in the same individual, which is much like the situation where some people develop allergic reactions in a given place, even though all people living in that area are exposed to the same external environment such as pollen in the spring. Additionally, the severity and the duration of allergic reactions also varies in these people. Another example is the cancer metastasis to the brain. Metastasis of cancer also varies from person to person based on the immune condition of patients. This is because; mast cell numbers and the extent of mast cell activation-mediated disease severity also varies from person to person.The brain was originally considered as an immunologically privileged organ but now it is well known that the peripheral immune system and the brain communicate through several pathways (Ransohoff and Brown, Regeneration of the neuronal tissue is limited following any CNS injury. The role of neurite outgrowth inhibitors such as Nogo proteins in inhibiting the neuro regeneration has been reported (Wang et al., Although the mechanisms of neuroinflammation mediated neurodegeneration is not yet clearly understood, neuroinflammation is considered an important contributing factor for the neuronal death in the brain of age-related neurodegenerative disease. Peripheral immune response/activation and peripheral inflammation could generate several proinflammatory and neuro cytotoxic mediators. These peripheral inflammatory factors as well as peripheral inflammatory cells such as mast cells and T cells enter the brain. Further, these cells and inflammatory mediators could directly or through astrocyte, microglia, neurons and mast cell activation induce and sustain chronic neuroinflammation leading to neuronal death in the brain. Although suppression of neuroinflammation could reduce the severity of disease symptoms and neurodegeneration, the aging factor and immune disorder could continue to influence neurodegeneration. Therefore, novel approaches are required to better understand the communication pathways between the systemic and CNS immune system for the development of new and effective strategies to limit neurodegeneration and promote neuroregeneration in the brain.DK and AZ wrote and critically edited the manuscript. RT, GPS, SZ, MEA, SPR, HZ, DS, PAN and SI edited 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.


Symbiodinium spp., from coral tissues) and subsequent coral mortality, devastating reefs worldwide. Genetic variation within and between Symbiodinium species strongly influences the bleaching tolerance of corals, thus recent papers have called for genetic engineering of Symbiodinium to elucidate the genetic basis of bleaching-relevant Symbiodinium traits. However, while Symbiodinium has been intensively studied for over 50 years, genetic transformation of Symbiodinium has seen little success likely due to the large evolutionary divergence between Symbiodinium and other model eukaryotes rendering standard transformation systems incompatible. Here, we integrate the growing wealth of Symbiodinium next-generation sequencing data to design tailored genetic engineering strategies. Specifically, we develop a testable expression construct model that incorporates endogenous Symbiodinium promoters, terminators, and genes of interest, as well as an internal ribosomal entry site from a Symbiodinium virus. Furthermore, we assess the potential for CRISPR/Cas9 genome editing through new analyses of the three currently available Symbiodinium genomes. Finally, we discuss how genetic engineering could be applied to enhance the stress tolerance of Symbiodinium, and in turn, coral reefs.Elevated sea surface temperatures from a severe and prolonged El Niño event (2014–2016) fueled by climate change have resulted in mass coral bleaching (loss of dinoflagellate photosymbionts,  Symbiodinium is considered one of the most important dinoflagellate genera given its role as the essential photosymbiont of many tropical reef invertebrates, notably reef-building corals  strongly influence coral gene expression and bleaching susceptibility  and a reporter gene (GUS) (Symbiodinium was reported (bar) and a reporter gene (GFP) were introduced to type A1, B1, and F1 strains using glass beads. Whilst cells transiently exhibited improved herbicide resistance and suggestive GFP signal, transformations were not validated through DNA, RNA, or protein analysis  to drive transcription of antibiotic resistance genes (ne (GUS) ; howeverreported . Plasmidanalysis . Furthero divide . Of thesSymbiodinium (and other dinoflagellates), we recommend a new approach that capitalizes on the recent surge in “omics” breakthroughs . By evaluating the rapidly increasing supply of next-generation sequencing (NGS) data, we propose a genetic engineering framework for Symbiodinium that may markedly advance our understanding of these important dinoflagellates. Furthermore, genetic manipulation of Symbiodinium in order to reduce coral bleaching has been hypothesized as a strategy to facilitate coral management as reefs continue to rapidly deteriorate under climate change , particularly the assembly of the first Symbiodinium genomes and transcriptomes, direct correlation between Symbiodinium transcriptional and physiological states, and discovery of genes from viruses actively infecting Symbiodinium cells. Furthermore, NGS of Symbiodinium has revealed genetic elements that may allow for transformation of Symbiodinium. In the following sections, we detail how unique Symbiodinium promoters, specific Symbiodinium genes underpinning important phenotypes, and a viral internal ribosomal entry site recognized by Symbiodinium ribosomes could be integrated to build expression constructs for Symbiodinium.Fundamental components of Symbiodinium , branch point (YTNAY), and acceptor for the dinoflagellate spliced leader (AG) (Symbiodinium promoters and terminators (as opposed to promoters and terminators from other organisms) would likely improve expression and stability of transgenes introduced into Symbiodinium. By chance, the CAMV 35S  promoter happens to contain all of the described Symbiodinium promoter elements, and the CAMV 35S  and nos (plant) terminators both contain the dinoflagellate polyadenylation signal; this may have contributed to their ability to drive transgene expression in Symbiodinium previously  . Additioder (AG) . Hence, eviously .Symbiodinium nuclear genes that can be genome-mapped to uncover strong, endogenous promoters and their corresponding terminators. These promoters and terminators can be isolated from purified genomic DNA (gDNA) through PCR and incorporated into custom DNA expression constructs for Symbiodinium . Among the most highly expressed transcripts in Symbiodinium transcriptomes are genes for peridinin-chlorophyll a-binding protein, caroteno-chlorophyll a-c-binding protein, major basic nuclear protein 2, dinoflagellate viral nucleoprotein, and glyceraldehyde-3-phosphate dehydrogenase  genes are believed to minimize thermally induced oxidative damage to photosynthetic apparatuses and leakage of harmful reactive oxygen species from type C1 Symbiodinium cells—determinants of coral bleaching  reverse transcribed from purified mRNA, since gDNA introns may prevent proper expression in constructs .Recent transcriptomic studies have been fundamental in the discovery of ral host . For insleaching ; howevervariants . A Fe-soSymbiodinium could also greatly advance investigations of ecological processes central to coral reef health. For instance, documenting competition between Symbiodinium types, transmission and acquisition of Symbiodinium types by the coral host, and shuffling of Symbiodinium types within host tissues , and 2A peptides are staple regulatory elements incorporated in expression constructs since they have evolved to be recognized by eukaryotic machinery for efficient and stable foreign gene expression . Symbiodiodinium . A putatcriptome , and sucFigure 2). Conversely, in monocistronic constructs, the selectable marker gene often maintains expression, while the gene of interest becomes transcriptionally repressed over time if it does not increase fitness of the cell  can be combined with the backbone of a standard cloning plasmid; added into an artificial, replicating minicircle  rather than RNA polymerase II. Therefore, promoters specifically recognized by Pol III  are needed. Such promoters have been isolated from other eukaryotes for sgRNA transcription; but, as discussed earlier, they contain motifs  that Symbiodinium lack  then finally the plasma membrane  was developed  can have negative impacts on a number of coral fitness traits including growth and fecundity . Based on these discoveries, we have developed a tailored genetic engineering framework for Symbiodinium based on empirical data that may also be applicable to other dinoflagellate genera. In doing so, we have opened a new prospective avenue to decode Symbiodinium functional genomics that may ultimately allow for engineering increased stress tolerance of Symbiodinium to reduce coral bleaching.As we have discussed here, recent NGS breakthroughs have revealed natural genetic elements of RL conceived the manuscript concept, analyzed NGS data, and wrote the manuscript. CRV analyzed NGS data and critically edited the theory and writing of the manuscript. SA analyzed NGS data. PS, DS, and MvO critically edited the theory and writing of 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 AWL declared a shared affiliation, though no other collaboration, with one of the authors DS to the handling Editor, who ensured that the process nevertheless met the standards of a fair and objective review.


Genetic testing is increasingly used for clinical diagnosis, although variant interpretation presents a major challenge because of high background rates of rare coding-region variation, which may contribute to inaccurate estimates of variant pathogenicity and disease penetrance.To use the Exome Aggregation Consortium (ExAC) data set to determine the background population frequencies of rare germline coding-region variants in genes associated with hereditary endocrine disease and to evaluate the clinical utility of these data.Cumulative frequencies of rare nonsynonymous single-nucleotide variants were established for 38 endocrine disease genes in 60,706 unrelated control individuals. The utility of gene-level and variant-level metrics of tolerability was assessed, and the pathogenicity and penetrance of germline variants previously associated with endocrine disease evaluated.In silico variant prediction tools demonstrated low clinical specificity. The frequency of several endocrine disease‒associated variants in the ExAC cohort far exceeded estimates of disease prevalence, indicating either misclassification or overestimation of disease penetrance. Finally, we illustrate how rare variant frequencies may be used to anticipate expected rates of background rare variation when performing disease-targeted genetic testing.The frequency of rare coding-region variants differed markedly between genes and was correlated with the degree of evolutionary conservation. Genes associated with dominant monogenic endocrine disorders typically harbored fewer rare missense and/or loss-of-function variants than expected. Quantifying the frequency and spectrum of rare variation using population-level sequence data facilitates improved estimates of variant pathogenicity and penetrance and should be incorporated into the clinical decision-making algorithm when undertaking genetic testing. The frequency of germline coding-region variation was evaluated in 38 endocrine disease genes in 60,706 individuals. The clinical utility of these data in improving genetic diagnosis is demonstrated. IncreasTherefore, to address these challenges, we used the ExAC cohort to quantify the spectrum and frequency of rare germline missense and loss-of-function (LOF) SNVs in 38 genes associated with hereditary endocrine disease and explored the utility of these data when applied to several clinical settings. Our results demonstrate the value of large control cohorts such as ExAC and illustrate how estimates of cumulative rare variant frequencies, together with additional gene- and variant-level factors, may be incorporated into the workflow for clinical genetic testing.http://exac.broadinstitute.org; accessed March 2016 to October 2017). Details of the contributing populations, sequencing methods, and variant filtering and calibration methods have been reported  constraint metrics were obtained directly from the ExAC browser . Detailed descriptions of these metrics are reported elsewhere, and a brief overview is provided in the Supplemental Appendix  were evaluated in the ExAC cohort. Disease-associated mutations were identified from publicly available sources (details are provided in delines) . For eacAIP SNVs with an AF <0.5% were established for the ExAC cohort and compared with those observed in 1866 individuals with apparently sporadic pituitary tumors reported in nine earlier studies. Odds ratios with 95% confidence intervals were established.Individual and cumulative frequencies of all missense Four disease-targeted gene panels were formulated to model estimated background frequencies of rare missense and LOF SNVs when undertaking multiple-gene sequencing. These represented PPGL, calcium-/parathyroid-related disorders, pituitary tumor disorders, and MEN syndromes. Cumulative rare variant frequencies were used to establish the likelihood that a given individual would harbor a rare variant in at least one of the panel genes.i.e., single-nucleotide substitutions resulting in missense or nonsense amino acid changes or directly affecting donor or acceptor splice sites) in each of the 38 genes revealed that the overwhelming majority were rare, with ~60% occurring as singletons , whereas ~92% of gene-specific SNVs had an AF <0.05%  (chromodomain helicase DNA-binding protein 7 (CHD7) and neurofibromin (NF1) demonstrated the highest frequencies of singleton variants , whereas the lowest frequency was observed for adaptor-related protein complex 2 sigma 1 subunit (AP2S1) . Of note, removal of the TCGA subgroup (n = 7601) from the analysis had a minimal impact on cumulative rare SNV frequencies of the genes evaluated , with the exception of von Hippel-Lindau (VHL), for which reduced sequence coverage of part of the gene reduced the reliability of the estimates .Thirty-eight genes were selected for study, representing a range of endocrine disorders reported to be associated with heterozygous germline missense and/or LOF SNVs . The ideviduals) . The cumviduals) . For exae.g., for singleton SNVs2, r= 0.84; P < 0.0001) , marked differences persisted after correcting for gene size . Furthermore, a significant correlation was observed between rare nonsynonymous SNV frequency and the degree of amino acid sequence identity between orthologs of the encoded protein, with the most highly conserved genes  demonstrating the lowest rates of rare variation .Although a positive correlation was observed between rare nonsynonymous SNV frequency and coding-region nucleotide length  . Of the 38 genes, 20 (53%) were categorized as missense and/or LOF intolerant  or X-linked‒dominant disorders , thereby indicating appropriate constraint against nonsynonymous heterozygous variation. In contrast, many disease-associated genes were categorized as missense and/or LOF tolerant, including several in which the role of heterozygous variation and endocrine disease is less established  or was previously associated with reduced disease penetrance . For other genes in the tolerant groups , their small size reduced the reliability and utility of the respective constraint metrics , the cumulative estimates of LOF SNV allele frequencies were established for each gene . Although many genes displayed an absence or very low number of LOF SNV alleles consistent with their known haploinsufficiency function , some genes harbored cumulative LOF SNV frequencies considerably higher than the associated disease phenotype , indicating a reduced penetrance of such variants. For other genes, , the apparent high LOF SNV frequency observed was consistent with the known disease prevalence . Similarly, small indels resulting in an LOF  were absent or very rare in the majority of genes , although higher frequencies were observed in several genes in which LOF indels would be anticipated to be disease causing, including NF1 , CASR , SDHB , and CDC73  . However, it is important to note that the reliability of indel variant calls is most likely reduced compared with SNVs, whereas larger indels will not be identified by capture-based sequencing methods.To further quantify the variability in LOF SNV frequency . To assess the potential utility of such tools, we analyzed SIFT, Polyphen2, and CADD scores of all rare missense SNVs in a subset of 12 genes. In total, an average of 21% of all rare SNVs in each gene were categorized as deleterious using criteria encompassing all three tools, whereas 53% were described as possibly deleterious . This analysis revealed that for several genes, the frequency of alleles reported as pathogenic far exceeded the reported prevalence of the associated disease , giving rise to four possible explanations: The prevalence of disease is higher than reported; prior reports of SNV pathogenicity are incorrect ; the gene- or allele-specific disease penetrance is lower than reported; or the cohort is unknowingly enriched for hereditary endocrine disorders. For example, approximately one in 1750 individuals was observed to harbor a pathogenic ret proto-oncogene (RET) allele , in which approximately one in 3500 individuals harbored a variant previously associated with FHH type 1 , although in this setting it is plausible that the condition is more prevalent than currently recognized because of its typically asymptomatic phenotype [MEN1 and VHL variants in the ExAC cohort indicates that several of these variants were likely misclassified. However, it is important to note that in the case of VHL, four of the six likely pathogenic variants occurred in individuals from the TCGA cohort, which included 344 patients with a history of sporadic renal clear cell carcinoma who may be at an increased risk of harboring such germline variants .An apparent similar overrepresentation of disease alleles was observed for the calcium sensing receptor . Of note, this collection of disease-associated SNVs did not demonstrate a significant excess of alleles originating from the TCGA cohort (P > 0.1) .Next, the ExAC cohort was evaluated for individuals harboring clinically-actionable variants in one of seven hereditary endocrine tumor predisposition genes currently included in the ACMG guidelines . Only prAIP are reported in familial isolated pituitary adenoma kindreds but are associated with reduced penetrance, making it difficult to differentiate between hereditary and sporadic forms [AIP variants have also been reported in individuals with apparent sporadic pituitary tumors, although ascribing pathogenicity may be challenging because several AIP variants are observed in both disease and control populations [AIP in this setting, we compared the frequencies of rare germline missense AIP variants in 1866 individuals with sporadic pituitary adenomas  with those observed in the ExAC cohort. Prior analysis demonstrated the predicted frequency of likely cases in the ExAC control population to be negligible . Of note, only a small excess of rare missense AIP variants was observed in the tumor cohort compared with the ExAC cohort , whereas no overall excess was identified when compared with the European ExAC subpopulation, selected to represent the most relevant cohort for comparison  were observed in the tumor group, indicating that such variants are most likely benign or associated with very low penetrance . A small but notable excess of novel singleton SNVs was observed in the tumor group, predominantly in patients with acromegaly, suggesting genuine enrichment of pathogenic variants in this subgroup . Thus, modeling of four disease-targeted gene panels  revealed high cumulative estimates of identifying rare variants for each panel , with ~3% of control individuals harboring a novel singleton variant . Such estimates were then modified to allow direct comparison with literature reports. For example, a recent study evaluating 14 genes in patients with sporadic PPGL reported a rare germline mutation frequency of 7%, which did not exceed the background frequency predicted from our analysis of the ExAC cohort [Finally, the gene-level estimates of rare SNV frequency were used to model expected rates of background variation likely to be observed when performing simultaneous sequence-analysis of multiple genes . Therefore, these genes are likely to be under strong evolutionary selection pressure to conserve key cellular processes, thereby resulting in a relative intolerance to variation.In this study, we used the ExAC cohort to quantify the spectrum and frequency of rare nonsynonymous germline variants occurring in a broad range of genes associated with hereditary endocrine diseases and illustrate the potential utility of this information for improved variant interpretation, both in ascribing potential pathogenicity and in reevaluating estimates of disease penetrance. Of note, we observed marked differences in the frequency or rare nonsynonymous SNVs between genes after correcting for coding-region length, and this was correlated with the degree of evolutionary conservation. The observation that the lowest rare SNV frequencies were observed in genes with the highest degrees of evolutionary conservation is not unexpected, as several of the most highly conserved genes regulate essential cellular functions , current analyses often use a combination of variant-level features, such as the absence of a variant in a control population, as well as predictions from computational tools [i.e., enabling categorization only as a variant of uncertain significance), they are frequently cited as supporting evidence of pathogenicity and may ultimately result in a patient being managed as if the variant is disease causing. Our analyses indicate that variant rarity  together with computational prediction tools frequently have a low specificity for ascribing clinically relevant effects and that relying on such features likely overestimates pathogenicity. For example, we observed that across the 38 genes, the majority of individual variants were observed only once in the ExAC cohort and that a similar majority of rare SNVs were classified as potentially deleterious by at least one of the computational tools evaluated. However, our studies also demonstrated how the use of additional gene-specific factors, including cumulative rare variant frequencies together with metrics of missense and LOF constraint, may provide important additional context when incorporated into the variant interpretation workflow . Indeed, it is plausible that in the future such gene-specific constraint metrics and/or estimates of rare variant burden may be incorporated into bioinformatic or clinical computational algorithms to improve estimates of variant pathogenicity and/or disease penetrance.Although consensus guidelines have been established by the ACMG for the clinical interpretation of germline sequence variants, in many instances an unambiguous assignment of pathogenicity is not possible . In the al tools . Althougworkflow . For exaFurthermore, we demonstrated how quantifying the burden of rare variation in a given set of genes may be used to derive an expected frequency of background rare variants that should be anticipated when using disease-targeted gene panels. Of note, these studies illustrate how a failure to consider the high frequency of background rare variation arising from the use of such gene panels is likely to contribute to diagnostic uncertainty and may have confounded earlier genetic studies.e.g.,RET, VHL, and MEN1), indicating their likely prior misclassification and/or overestimates of disease penetrance. Indeed, the need to define allele-specific estimates of disease penetrance is an important concept, as recently illustrated for prion disease in which the disease-penetrance of individual PRNP variants ranged from <0.1% to ~100% [e.g.,RET), and quantifying these is essential to enable appropriate patient care  in a large pituitary tumor cohort compared with the ExAC population cannot exclude an etiological role in disease, although it suggests that any disease relationship is associated with extremely low penetrance and that the overwhelming majority of such variant carriers will not manifest clinical features. Indeed, establishing accurate estimates of disease penetrance should be a priority, and it is evident that for several hereditary endocrine genes, penetrance may have been overstated  because of unintentional ascertainment bias and/or the inclusion of index cases in such estimates [However, differentiating benign from low-penetrance alleles remains challenging, even with large disease and control cohorts . For exastimates .e.g., the presence of founder mutations in local populations). For example, although ExAC offers large numbers of individuals from specific ethnic groups , other populations are underrepresented, and for these groups, it may not provide a suitable comparator group.However, for accurate estimates of penetrance to be established, it is essential that the control population used be closely matched to the study population, thereby avoiding confounding from population-specific differences in variant frequencies  [e.g., in SDHB and SDHD). This not only highlights the potential clinical burden that increased genetic testing may bring but also emphasizes the need for accurate estimates of variant pathogenicity and penetrance because the potential for patient harm arising through tumor surveillance programs is not insignificant [Periodic clinical, biochemical, and/or radiological screening is generally recommended for carriers of pathogenic alleles associated with hereditary endocrine tumor syndromes (nd SDHB) , 27, 29.nificant , 30. Fure.g., PPGL, medullary thyroid cancer), it was reassuring that the TCGA samples did not contribute an excess of rare SNVs or likely pathogenic alleles in the genes investigated, with the possible exception of VHL, in which the inclusion of renal carcinoma cases may have introduced a risk of bias.Our analysis has several potential limitations. First, although not knowingly enriched for hereditary endocrine disorders, the ExAC cohort will contain individuals with these diseases as well as controls with polygenic disorders with accompanying disease-associated alleles. However, the inclusion of a broad range of genes and associated conditions reduced the likelihood of widespread enrichment for relevant disease phenotypes. For example, one potential concern was that the inclusion of germline samples from the TCGA cohort might result in an overrepresentation of alleles in genes associated with hereditary endocrine tumors; although this cohort does not include endocrine tumors relevant to the genes under study  could not be evaluated. In addition, high-quality SNV calls were assumed to be accurate, and although the performance of the ExAC variant calling pipelines has been extensively validated , confirmatory sequencing of individual variants was not undertaken. Furthermore, caution is required in interpreting SNVs in regions with reduced sequence coverage  or those presenting difficulties in the sequencing pipelines. For example, the reliability of sequence data for genes with multiple pseudogenes  may be reduced, although in these instances, visual inspection of individual sequence reads covering regions adjacent to the SNVs enabled increased confidence in the variant call .A further limitation of the current study is that individual rare variants were considered in isolation in our analysis, and potential interactions between variants or other modifying influences .Another limitation of our study is that we excluded indels from the main analysis because of their reduced reliability of detection ; althougIn summary, these studies demonstrate how quantifying rare germline variations in a large control cohort such as ExAC may be exploited to improve variant interpretation and clinical decision-making. Furthermore, this information may be incorporated into different stages of the clinical genetic testing workflow  and may


Contemporary advances in addiction neuroscience have paralleled increasing interest in the ancient mental training practice of mindfulness meditation as a potential therapy for addiction. In the past decade, mindfulness-based interventions (MBIs) have been studied as a treatment for an array addictive behaviors, including drinking, smoking, opioid misuse, and use of illicit substances like cocaine and heroin. This article reviews current research evaluating MBIs as a treatment for addiction, with a focus on findings pertaining to clinical outcomes and biobehavioral mechanisms. Studies indicate that MBIs reduce substance misuse and craving by modulating cognitive, affective, and psychophysiological processes integral to self-regulation and reward processing. This integrative review provides the basis for manifold recommendations regarding the next wave of research needed to firmly establish the efficacy of MBIs and elucidate the mechanistic pathways by which these therapies ameliorate addiction. Issues pertaining to MBI treatment optimization and sequencing, dissemination and implementation, dose–response relationships, and research rigor and reproducibility are discussed. Advances in biobehavioral science occurring over the past several decades have made significant headway in elucidating mechanisms that undergird addictive behavior. This large body of research suggests that addiction is best regarded as a cycle of compulsive substance use subserved by dysregulation in neural circuitry governing motivation and hedonic experience, habit behavior, and executive function . Though Contemporary developments in addiction neuroscience have been paralleled by increasing interest in the age-old mental training practice of mindfulness meditation as a potential treatment for addictive behavior. This interest was sparked by the successful integration of mindfulness techniques into secularlized behavioral intervention programs, including Mindfulness-Based Stress Reduction (MBSR)  and Mindstate of mindfulness—i.e., a state of metacognitive awareness characterized by an attentive and nonjudgmental monitoring of moment-by-moment cognition, emotion, sensation, and perception without perseveration on thoughts of past and future. The practice of mindfulness has been proposed to involve two primary elements: focused attention and open monitoring [Derived from ancient Indo-Sino-Tibetan contemplative practices and philosophies concerning the cultivation of awareness, the construct of mindfulness has been alternately operationalized as a state, trait, and practice in the modern scientific literature. MBIs provide training in practices designed to evoke the nitoring , 8. Durinitoring . Open modispositional or trait mindfulness) even when one is not engaged in meditation practice [mindlessness [Frequent and regular practice  of mindfulness techniques is thought to cultivate durable changes in the trait-like propensity to be mindful in everyday life  for addiction were modeled after the first generation of mindfulness-based therapies like MBSR and MBCT in terms of their structure and format. MBIs for addiction tend to be multi-week interventions (approximately 8 weeks in duration) usually delivered in a group therapy format. Each week, participants are guided by a trained clinician in various mindfulness practices, including mindful breathing and body scan meditations. These in-session mindfulness practices are debriefed during a subsequent group process, after which new psychoeducational material is typically presented. Sessions often involve experiential exercises to reinforce the mindfulness principles that had been introduced didactically. Participants are given therapeutic homework, consisting of formal and informal mindfulness practices as well as assignments to self-monitor symptoms like craving and negative affect. Extant MBIs for addiction differ from one another in terms of the types of mindfulness practices taught, the style in which these practices are delivered and debriefed , the length of at home mindfulness practice sessions, and the specific psychoeducational content delivered.MBIs for addiction are usually tailored to address pathogenic mechanisms implicated in addiction by targeting mindfulness techniques to addictive behaviors  and by discussing the application of mindfulness skills to cope with addiction in everyday life. For instance, MORE participants are guided to engage in the “chocolate exercise”— an experiential mindfulness practice designed to increase awareness of automaticity and craving . During In a mechanistic theoretical account of mindfulness as a treatment for addiction, Garland, Froeliger, & Howard conceptualized MBIs as means of mental training designed to exercise a number of neurocognitive processes that become dysregulated during the process of addiction . Such meFrom this perspective, MBIs can been seen as behavioral strategies for strengthening the integrity of prefrontally-mediated cognitive control networks that have become atrophied by chronic drug use and hijacked by drug-related cues and cravings during the process of addiction. As adaptive cognitive control is restored through mindfulness exercises, MBIs may increase functional connectivity between these top-down prefrontal networks and bottom-up limbic-striatal brain circuitry involved in reward processing and motivated behavior . Increasthe restructuring reward hypothesis [savoring [Through mechanistic effects of mindfulness-induced enhancements in functional connectivity between top-down and bottom-up brain circuitry, MBIs may reverse the allostatic process by which normal reward learning is usurped by addictive substances. In that regard, Garland recently advanced a novel hypothesis concerning the therapeutic mechanisms of mindfulness for treatment of addiction: pothesis . The respothesis  and prompothesis . Indeed,pothesis , 27, andpothesis . This apsavoring .restructuring reward hypothesis and suggests that increasing natural reward processing through MBIs might reduce craving and addictive behavior. In mechanistic analyses from a RCT of MORE as a treatment for prescription opioid misuse among chronic pain patients, MORE produced significant increases in cardiac-autonomic and electrocortical responses to natural reward stimuli that were, in turn, associated with decreases in opioid craving [Though the aforementioned studies were not directly concerned with substance use disorder treatment, a mounting body of evidence supports the  craving , 31, sug craving . These p craving .restructuring reward hypothesis were paralleled by preliminary functional magnetic resonance imaging (fMRI) evidence of the effects of MORE on nicotine dependent smokers. In a pilot study of MORE as a smoking cessation intervention [These findings supporting of the rvention , smokersBy strengthening top-down cognitive control, MBIs may improve executive functions like self-control over automatic habits, decision-making, and response inhibition that are crucial to reducing drug use and maintaining abstinence. In that regard, a small quasi-experimental study of a mindfulness and goal management training intervention for polysubstance use demonstrated significant improvements in executive functioning, including working memory, selective attention/response inhibition and decision-making skills following mindfulness training relative to treatment as usual . A subseMBIs might also reduce addictive behavior by strengthening facets of dispositional/trait mindfulness. In a RCT of MBRP among a heterogenous sample of individuals with various substance use disorders, increases in dispositional mindfulness facets like acceptance, awareness, and nonjudgment significantly mediated the effect of MBRP on decreasing craving following treatment . SimilarGiven known linkages between stress and addiction , 43, MBIMBIs may reduce addictive behavior by decreasing subjective craving and attentional and physiological indices of drug cue-reactivity. In addition to the aforementioned smoking cessation study in which MORE reduced striatal responses to cigarette cues , decreasFinally, given that suppression of addictive urges exhausts autonomic resources for self-control  and paraThough mechanistic research on MBIs has begun to amass, there are few psychophysiological and neuroimaging studies of MBIs as a treatment for addiction. Thus little data exists to either support or refute the neural mechanistic models proposed in this section. Clearly, more research is needed in this area.d = 0.33, − 0.49 to 0.17, p < 0.05), cigarette smoking , craving , and stress  domains.A considerable body of findings has amassed supporting the capacity of MBIs to reduce substance use and attenuate factors promoting substance use, such as craving and stress. Over the past decade, multiple systematic reviews have been conducted to identify the effects of MBIs on addictive behaviors, and have found accumulated evidence for the positive effects of MBIs –63. Mored = 0.62, − 0.02 to 1.26, p = 0.057). In individual studies, MBIs produced a host of other significant salutary effects including increases in emotion regulation [With regard to secondary or mechanistic outcomes, as expected, MBIs produced significant increases on the Five Factor Mindfulness Questionnaires in all eight studies that used this measure .Of the 34 RCTs reviewed in this meta-analysis, ten used treatment-as-usual comparison groups, whereas two used inert comparison groups, sixteen employed an alternative psychotherapeutic treatment , and six examined brief mindfulness treatments compared to alternative therapies in laboratory settings. Twenty-eight of the reports presented the first published findings from the related study and six reports presented results of secondary analyses. Any given study could contribute findings only once to meta-analyses conducted within outcome domains. The adequacy of randomization was examined in all studies and analysis of covariance and linear mixed modeling were often used to control for any remaining pretreatment differences. Nearly half of the studies had samples sizes less than fifty. Many studies had high attrition rates at posttreatment and subsequent follow-ups. Most of the 34 studies reviewed relied extensively on self-report measures of substance use and other constructs. All RCTs examined were single-site studies. The most common methodological limitations were failure to interview collateral informants regarding study participants’ substance use behaviors at posttreatment and follow-up and to employ posttreatment and follow-up interviewers who were blind to participants’ treatment assignments. Fewer than half of the RCTs employed objective verification of participants’ self-reported substance use, such as urinanalysis.Subgroup analyses within outcome domains indicated that MORE treatment was associated with larger effects than other MBIs for substance use, craving, stress, and mindfulness measures . StudiesLi et al.  also repRandomized controlled trials suggest that MBIs are a promising treatment for substance misuse and exert their effects via increases in levels of mindfulness across a wide array of substance-misusing behaviors and clinical populations. Future research should employ larger samples, longitudinal designs with follow-up periods of at least 1-year, manualized interventions with treatment fidelity assessment, intent-to-treat analyses, and probability sampling designs allowing generalizability to specific clinical and general populations.MBIs are promising treatments for addiction. Results from rigorous, full-scale RCTs indicate that MBIs can produce short and long-term reductions in craving and addictive behavior. At this juncture in the development of the field, additional Stage III and IV clinical trials  are neemediation (“How do MBIs improve addiction-related outcomes?”) and moderation . As discussed in “Assuming an affirmative answer to the aforementioned question, studies should then aim to address research questions pertaining to A number of additional research questions remain unanswered. Here we lay out an agenda for the next wave of research in the field.restructuring reward hypothesis . Furthermore, molecular neuroimaging  is needed to understand effects of MBIs on neurotransmitters and neuropeptides implicated in addictive behavior like dopamine, endogenous opioids, γ-aminobutyric acid (GABA), and endocannabinoids. Finally, dynamic effects of mindfulness practice on addictive responses are unknown, and could be elucidated through functional neuroimaging techniques with high temporal resolution like electroencephalography (EEG) or magnetoencephalography (MEG). Such methods could answer other pertinent questions. For instance, does the acute state of mindfulness attenuate initial attentional orienting to drug cues? Or, does mindfulness facilitate attentional disengagement and recovery from drug cue-exposure? These questions can be answered by investigating how mindfulness training influences the time course of neural responses to drug cues.Little is known about the neurobiological mechanisms of mindfulness as a treatment for addiction. Though various conceptual models have been advanced , 23, 73,Although understanding treatment mechanism is not necessary to establish a given treatment modality as an empirically supported intervention, understanding the mechanisms of mindfulness can inform the refinement of MBIs to yield larger effect sizes and produce additional therapeutic benefits. A case in point is MORE, which was refined based on mechanistic discoveries. Following the first trial of MORE, it was found that mindfulness reduces pain severity by fostering a shift from affective to sensory processing of pain as innocuous sensory information . As a reIt is not known whether MBIs are most efficacious as standalone treatments or as a part of a more comprehensive treatment package. In many inpatient addictions treatment programs, clients receive multiple behavioral interventions  during the same 30-day time frame. Further, optimal treatment sequencing has not been studied. For instance, would MBIs be more efficacious following several sessions of motivational interviewing? Given that MBIs involve mindfulness practice, and regular practice requires motivation, introducing several sessions of motivational interviewing before initiating a course of mindfulness training might increase practice engagement and thereby boost clinical outcomes. Conversely, mindfulness training might potentiate motivational enhancement therapy by increasing interoceptive awareness of adverse consequences of addictive behavior on bodily health. In a similar vein, mindfulness training might increase adherence to medication-assisted therapy (MAT) by increasing awareness of how medication adherence allays the dysphoria associated with craving and thereby potentially improves quality of life. In turn, MAT might improve adherence to MBIs by attenuating distracting withdrawal symptoms and decreasing obsessive thinking about obtaining the next drug dose, thereby freeing cognitive and motivational resources to devote to learning mindfulness skills. Psychopharmacological interventions, cognitive training via computer- or smartphone-deployed technology, neurofeedback, and neurostimulation  administered prior to initiating a course of MBI might also improve cognitive function to facilitate learning of mindfulness techniques, and thereby improve MBI outcomes.Sequential, multiple assignment, randomized trials (SMART) could be used to assess the efficacy of dynamic treatment regimens, including those that are individually tailored based on decision rules that dictate how the type or dosing of treatment should change based on the specific clinical needs of the patient . For insIn pharmacological research, it is imperative to examine dose–response relationships to identify the optimal therapeutic dose. Dose–response curves can help to identify the dose needed to achieve a satisfactory clinical outcome while minimizing the side-effect profile of the drug. Although MBIs delivered in clinical settings appear to have few adverse effects , the cosGiven meta-analytic findings that extent of mindfulness practice is significantly associated with treatment outcomes , differeOne of the greatest challenges confronting the movement towards evidence-based practice in addictions treatment is the research-to-practice gap: that is, empirically-supported therapies with proven efficacy as revealed by Stage II randomized clinical trials are often not successfully translated into effective clinical interventions in standard addiction practice settings . SuccessIn outlining issues pertaining to advancing the clinical science of MBIs, Dimidjian and Segal highlight the tension between the need to make MBIs disseminable in the context of real-world resource constraints and complex client populations while not allowing outcomes to suffer as MBIs are scaled up in the translation to community treatment settings . This isFinally, it is unknown whether mindfulness might best ameliorate addiction through participation in time-limited interventions or if mindfulness should be used daily as part of a wellness lifestyle. With regard to the latter, shifting from an addiction-oriented lifestyle to adoption of a wellness lifestyle is conceptualized as integral to the recovery model . In thisFrom a neurobiological perspective, increasing grey matter density, strengthening of white matter tracts, synaptic remodeling, and other neuroplastic modifications to brain structure and function needed to undo the pathophysiology of addiction might require recurrent mindfulness practice for the long-term. From a psychological perspective, long-term mindfulness practice may be needed to induce self-referential plasticity and facilitate flexible reconfiguration of the self-schema in relation to the world  so as tod = 0.68) and substance misuse (pooled Cohen’s d = 0.33), suggesting that MBIs may be efficacious treatments for addiction. Overall, a number of RCTs with active control conditions have been conducted in the past decade—a sign that the methodological rigor of this field is increasing. However, with several notable exceptions , 92d = 0Coming full circle, MBIs are some of the newest additions to the armamentarium of addictions treatment. It is perhaps no coincidence that the rise of MBIs has been co-incident with advances in the neuroscience of substance use disorders. In recognizing that addiction is, in large part, mediated by cognitive and behavioral automaticity propelled by alterations to hedonic regulatory systems in the brain, this perennial form of human suffering may be especially tractable to treatment approaches like mindfulness that enhance top-down conscious control over bottom-up automatic habits and motivational drives. Insofar as the original purpose of many mindfulness meditation practices was to extinguish craving by revealing the “middle way” between attachment to pleasure and aversion to pain, MBIs may ultimately provide a skillful means of liberating the individual from the push and pull of hedonic dysregulation underlying addiction.


Yang et al. present a comprehensive overview of hair cell damage across stimuli. Other papers in this Research Topic offer more focused reviews or original research, as described below.Sensory hair cells are specialized mechanosensory receptors found in vertebrate auditory, vestibular and lateral line organs that transduce vibratory and acoustic stimuli into the sensations of hearing and balance. Sensorineural hearing loss typically occurs when hair cells are damaged from infection, noise exposure, drugs , and age-related decline. Unfortunately, as hair cell regeneration does not occur to any significant extent in mammals, damage to these cells in humans leads to hearing and balance disorders. The signaling mechanisms that cause hair cell damage are incompletely understood and vary with the type of damage involved, and a given form of damage can activate multiple cell signaling cascades, complicating our understanding of these damage processes and the development of potential therapeutics. Van Rossom et al. investigated programmed cell death resulting from variants in DFNA5, a gene tightly linked to autosomal dominant hearing loss in human families. DFNA5 is located on chromosome 7 and multiple variants are correlated with deafness in different human families. Using yeast, Van Rossom et al. demonstrate that truncated DFNA5 (mutDFNA5) alters expression of mitochondrial and endoplasmic reticulum (ER)-related genes, while mutDFNA5 expression in HEK293 cells up-regulated expression of MAPK signaling members. MutDFNA5 expression increased phospho-JNK expression in HEK cells and reduced cell viability, which could be rescued by pharmacological inhibition of JNK signaling. This suggests that mutDFNA5 induces cell death via oxidative stress mechanisms associated with mitochondrial function, and perhaps via ER stress, via MAPK signaling.Several papers in this Frontiers Research Topic address cell signaling that underlies hair cell damage from a variety of sources. While genetic causes of hearing loss accounts for the majority of congenital deafness, ototoxin exposure can cause hearing loss later in life. Aminoglycoside antibiotics are used to treat a wide-variety of bacterial infections, and up to 25% of patients receiving aminoglycosides suffer significant hearing loss. Aminoglycoside ototoxicity is extensively studied, because these antibiotics are well characterized, relatively simple to administer in animal models, and produce dose-dependent sensory tissue lesions. Multiple papers in this Research Topic investigate aspects of aminoglycoside-induced hair cell death and protection.Tao and Segil used RNA-Seq to profile transcriptional changes in hair cells exposed to the aminoglycoside gentamicin. They cultured neonatal cochleae from Atoh1-GFP transgenic mice, then purified GFP+ hair cells for sequencing and bioinformatics analysis. A 3 h exposure to gentamicin changed gene expression in several cell death and survival pathways, particularly JNK and NF-kB, and in several cell cycle checkpoint and proliferation genes. The effects on these latter pathways are interesting because hair cells are post-mitotic, and these data suggest that gentamicin treatment may disrupt cell cycle control. As aminoglycosides also cause nephrotoxicity, Tao and Segil then compared their results with those from researchers who studied gentamicin-treated kidney cells and found similar changes in gene expression across multiple gene classes. Their results suggest that gentamicin-mediated damage in hair cells causes changes in JNK and NF-kB signaling that precedes cellular stress responses and programmed cell death.Sun et al. investigated cell death and survival genes in mouse cochleae treated with the aminoglycoside neomycin and found increased expression of pro-cell death genes such as Diablo, Htra2, and multiple caspases. Interestingly, these changes were most pronounced in young animals (P8-P14), implying a “sensitive period” in which cochlear hair cells are more susceptible to damage. Using a transgenic mouse line that over-expresses the pro-survival gene Xiap, Sun et al. demonstrated that increased Xiap expression reduced caspase levels in cochlear hair cells and conferred some protection from neomycin-induced hearing loss.Dinh et al. summarize several of these ROS studies, as well as highlighting the complexity of hair cell death signaling overall, including activation of both classical apoptotic and non-apoptotic components. Majumder et al. examined one component of the oxidative stress response, the ROS scavenger glutathione (GSH), during cochlear development and neomycin exposure. Using a fluorescent reporter, they showed that GSH levels increase with age in the mouse cochlea and that GSH expression does not change across the length of the cochlea, which is surprising as basal hair cells are more susceptible to damage than apical hair cells. Even more surprising, pharmacologic depletion of GSH did not sensitize cochlear hair cells to neomycin damage. These results add to the ongoing debate about the role of oxidative stress in aminoglycoside ototoxicity.Oxidative stress is a key mediator of hair cell death, with multiple studies reporting increased reactive oxygen species (ROS) following aminoglycoside exposure and antioxidant-related hair cell protection. Monroe et al.). The zebrafish lateral line is particularly useful for screening genes and compounds that affect hair cell responses to ototoxin exposure. Stawicki et al. review several of these studies, describing 3 novel mutations and over 20 compounds associated with reduced aminoglycoside susceptibility. Uribe et al. used the zebrafish lateral line as a model system to determine if pharmacologic activation of hepatocyte growth factor (HGF) signaling conferred protection from aminoglycoside toxicity. The HGF mimetic Dihexa protected hair cells from neomycin damage via activation of c-Met, the cognate HGF receptor, and conferred protection via Akt and MEK signaling.Other than mammalian models, zebrafish, a common model organism to study vertebrate development and gene function, is also used to study hair cell death and regeneration. Like other non-mammalian vertebrates, zebrafish have the capacity to regenerate lost sensory hair cells . Prior studies demonstrated that mechanotransduction (MET) channels at the stereociliary tips are the primary route of aminoglycoside entry into hair cells . Lin et al. found that Lgr5 was expressed in a subset of supporting cells in the striolar region of the neonatal mouse utricle following neomycin-induced hair cell loss. Further, they showed that Lgr5-positive supporting cells can regenerate new hair cells in explant cultures. Zhang et al. characterized the spatiotemporal expression of Lgr6 in the cochlear duct of embryonic and postnatal mice. They found that Lgr6 was first expressed in a single row of prosensory cells in the middle and basal turn of the cochlea, followed by expression being restricted to inner pillar cells (IPCs), and then the inner border cells (IBCs), after which expression disappeared. Wnt signaling was required to maintain Lgr6 expression in vitro and Lgr6-positive cells had the capability to differentiate into hair cells in culture. Thus, Lgr5- and/or Lgr6-postive cells may serve as hair cell progenitors in the mammalian cochlea.Knowledge gained from the role played by Wnt and Notch signaling in hair cell development has led researchers to investigate how the process of hair cell regeneration recapitulates developmental pathways. Two downstream target genes of the Wnt pathway are highlighted in this Research Topic: Tarang et al. recently developed a tetracycline-inducible TetO-CB-myc6-Rb1 (CBRb) transgenic mouse model to achieve transient and inducible dominant-negative inhibition of the protein retinoblastoma 1 (RB1), an endogenous protein essential in regulating cellular proliferation, differentiation, and homeostasis. Down-regulation of RB1 leads to increased proliferation and supernumerary hair cells in the cochlea, suggesting that RB1 may be a therapeutic target.Advances in biotechnology are improving ways that researchers can study molecular mechanisms that function in hair cell death and regeneration. For example, Jahan et al. review the plethora of genes involved in hair cell proliferation and differentiation in relation to the key transcription factor Atoh1, suggesting that a more nuanced view of Atoh1 regulation is necessary to explain potentially conflicting data. As support for point 2, chromatin remodeling, by histone modifications such as acetylation, has been shown to be important in cellular processes such as development and regeneration. Histone deactylases (HDACs) remove acetyl groups from histone tails, which represses transcription. Following hair cell ablation with neomycin, pharmacological inhibition of HDACs increased histone acetylation in zebrafish lateral line hair cells and decreased the number of proliferating supporting and regenerated hair cells . The underlying mechanism of HDAC regulation of hair cell regeneration is unknown, but pathways involved in HDAC or histone acetyltransferases (HATs) may be targets for therapeutics promoting hair cell regeneration. In support of this point, Layman and Zuo offer a review of epigenetic regulation in the inner ear in relationship to development, otoprotection, and hair cell regeneration.It is unlikely that the manipulation of one gene or the administration of a single compound will promote mammalian hair cell regeneration because (1) hair cell regeneration proceeds via multiple cellular pathways, and (2) epigenetic regulation controls these pathways. In support of point 1, Collectively, these papers highlight the recent progress in understanding the cellular and molecular processes involved in sensory hair cell death and regeneration. While there currently is no cure for sensorineural hearing loss in humans, recent experiments like those highlighted in this Research Topic provide a clearer picture of what genes and signaling mechanisms are involved and what therapies may prevent hearing loss, and potentially promote hair cell regeneration in the future.in situ supporting cells to induce transdifferentiation into new hair cells? While the use of stem cell-derived precursor cells show promise, technical difficulties include maintaining the survival of cell grafts in the proper locations along the cochlea without further damaging the cochlea, prevention of tumor formation, and potential graft-vs.-host rejection. Transdifferentiation of in situ supporting cells into hair cells is also difficult, as supporting hair cells represent a terminally differentiated state that is not overly amenable to manipulation. Since there are numerous molecular pathways involved in the development of sensory hair cells, researchers are not likely to find a single silver bullet in the form of a specific gene or protein that will restore hearing when manipulated or supplied therapeutically. Although supernumerary new hair cells have been induced in the mature mammalian cochlea, this has rarely led to any improvement in hearing function due to the precise organization of the rows of hair cells in the organ of Corti and the necessity of forming synaptic contacts between these hair cells and the appropriate spiral ganglion neurons . So far, this work has centered around a few ototoxic compounds such as the aminoglycosides gentamicin or neomycin. Since different ototoxic compounds can have different effects on hair cells, much more research is needed to get a more holistic view of how to prevent ototoxin-induced hair cell loss. For example, some evidence suggests that aminoglycoside antibiotics enter hair cells via the mechanotransduction channel , NIH R01DC014832 (to AG), and NIH R15DC013900 and an Action on Hearing Loss grant (to AC).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.