Both inactivated poliovirus vaccine (IPV) and oral poliovirus vaccine (OPV) have contributed to the rapid disappearance of paralytic poliomyelitis from developed countries despite possessing different vaccine properties. Due to cost, ease of use, and other properties, the Expanded Programme on Immunization added OPV to the routine infant immunization schedule for low-income countries in 1974, but variable vaccine uptake and impaired immune responses due to poor sanitation limited the impact. Following launch of the Global Polio Eradication Initiative in 1988, poliomyelitis incidence has been reduced by >99% and types 2 and 3 wild polioviruses are now eradicated, but progress against type 1 polioviruses which are now confined to Afghanistan and Pakistan has slowed due to insecurity, poor access, and other problems. A strategic, globally coordinated replacement of trivalent OPV with bivalent 1, 3 OPV in 2016 reduced the incidence of vaccine-associated paralytic poliomyelitis (VAPP) but allowed the escape of type 2 vaccine–derived polioviruses (VDPV2) in areas with low immunization rates and use of monovalent OPV2 in response seeded new VDPV2 outbreaks and reestablishment of type 2 endemicity. A novel, more genetically stable type 2 OPV vaccine is undergoing clinical evaluation and may soon be deployed prevent or reduce VDPV2 emergences. The conquest of polio is widely acknowledged to be one of the great scientific and medical achievements of the 20th century. The story is well known; periodic poliomyelitis epidemics first appeared in Scandinavia, Western Europe, and the United States in the late 1800s and steadily grew in frequency and magnitude until the 1950s and the early 1960s when inactivated poliovirus vaccine (IPV) and, later, live attenuated oral poliovirus vaccine (OPV) were introduced, leading to immediate and dramatic control of poliomyelitis in the United States and other industrialized countries [1]. In the United States, polio incidence fell from 13.9 cases per 100 000 in 1954 to <0.5 cases per 100 000 in 1965, endemic transmission ceased by 1970, and the last case of domestically acquired poliomyelitis was reported in 1979. Thereafter, only rare imported cases or cases caused by the live attenuated OPV  were observed, and today imported poliomyelitis has all but vanished with global control of polio and adoption of IPV-only schedules in many countries.However, in low-income countries with crowding and poor sanitation, recognition of the impact of poliomyelitis was long obscured by universal infection at an early age and the dogma that epidemic poliomyelitis was a disease of wealthier nations because exposure at older ages in countries with better hygiene increased the risk of paralytic disease. This misconception was rectified when many community- and school-based lameness surveys documented that approximately 1:100 to 1:200 children in poor countries suffered the paralytic consequences of poliomyelitis, rivaling the incidence rates observed in peak epidemic years in developed countries . Improvements in production technology in the 1970s, including the use of microcarrier cultivation to increase cell density, led to the introduction of “enhanced potency” formulations meeting a universal standard of 40, 8, and 32 D antigen units, for types 1, 2 and 3, respectively, per dose. The immune response to IPV depends on the number of doses, the interval between doses, and the presence of maternally derived antibody, which impairs immune responses in infants up to 6 months of age [5–7]. Detectable serum neutralizing antibody protects against disease, and a ≥4-fold rise in neutralizing antibody titer is widely accepted as the standard defining seroconversion [8]. When administered according to common recommendations, a 3-dose infant immunization schedule results in seroconversion rates of 85%–100% against each serotype . Successful field trials were carried out from 1955 to 1959 and OPV was introduced for routine use between 1961 and 1962 as sequentially administered monovalent vaccines. Trivalent OPV (tOPV) became available in 1964 and was rapidly adopted for infant immunization in all but a few countries due to superior immunogenicity compared with the IPV vaccines then available, lower cost, and ease of administration. Although rare cases of VAPP were recognized shortly after the introduction of OPV , tOPV reThe low cost, ease of administration, spread of vaccine virus to unimmunized, susceptible persons, and induction of gastrointestinal immunity make OPV the logical choice for continued use in low-income countries. For many years, tOPV was delivered for routine immunization through the EPI and via campaigns by the Global Polio Eradication Initiative (GPEI). After 2005, monovalent vaccines for type 1 (mOPV1) and type 3 (mOPV3), and later type 1 and 3 bOPV vaccine, were deployed in supplementary immunization campaigns in the remaining polio-endemic countries to overcome the problem of intertypic interference from the type 2 OPV in the trivalent vaccine and enhance seroconversion to types 1 and 3 –17]. TheA large study in India found that 2 bOPV doses at birth and 28 days of age induced seroconversion rates of 86% and 74% to type 1 and type 3 polioviruses, respectively, superior to respective rates of 63% and 52% observed with a comparator tOPV vaccine and noninferior to monovalent OPV1 and OPV3 vaccines . Similar6 birth cohort (250–500 cases) per year in tOPV-using countries [27],.VAPP is a rare, but serious complication of OPV use observed in OPV recipients and their contacts that is clinically indistinguishable from naturally occurring poliomyelitis. Prior to 2014, the global incidence of VAPP was estimated to be 2–4 cases/10ies [27],. Since tThe success of the smallpox eradication program in the 1970s led to the establishment of the EPI in 1974 when the public health community increasingly sought to harvest the full potential of vaccines. Rotary International had targeted poliomyelitis prevention as a priority and began to fund poliovirus vaccines for some of the poorest countries. In 1985, the Pan American Health Organization (PAHO) resolved to eliminate poliomyelitis from the Western Hemisphere. Rapid progress toward this goal encouraged the global public health community to consider whether poliomyelitis might be a candidate for a global eradication target. At that time, many policy makers had personal experience with children developing this crippling disease and were committed to prevent this illness, and technical reviews rated polio as the top candidate on the list of potential eradicable diseases.These developments led to a 1988 resolution by the World Health Assembly, the governing body of WHO, to eradicate poliomyelitis by the year 2000 . GPEI, cWhen the GPEI was established in 1988, >125 countries had an estimated poliomyelitis burden that totaled >350 000 cases annually. Implementation of core strategies initially developed by PAHO and adapted to polio-endemic countries in Asia and Africa, including acute flaccid paralysis surveillance, supplementary immunization campaigns, and support for routine infant tOPV immunization, led to a rapid decrease in global polio incidence, first in countries with better-developed health systems, including the Americas where the last indigenous WPV case was detected in 1991; the WHO Western Pacific Region, including China, where the last case was observed in 1997; and in Europe, where the same milestone was reached in 1999  [30–32].By the mid-1990s, eradication efforts were fully operational in all polio-endemic countries including India, where >50% of the world`s poliomyelitis cases were reported, and in sub-Saharan Africa, where a football (soccer)–themed initiative, “Kick Polio Out of Africa,” was launched in 1997 [33]. By 2000, although the original goal of eradication was not achieved, the number of countries reporting poliomyelitis had declined to 20 and the number of cases had decreased by >99%. Furthermore, the last indigenous wild poliovirus type 2 (WPV2) case was detected in northern India in 1999 [34].Rapid progress against type 1 and 3 polioviruses continued into the new millennium with a reduction of the number poliomyelitis cases from WPVs to a few hundred by 2010, all of which were confined to just 4 endemic countries . Eradication activities now focused increasingly on countries with suboptimal health systems in Asia, the Middle East, and Africa, where frequent importation of WPVs from the endemic countries, primarily type 1, created special challenges, as did recognition that the immunogenicity of tOPV was low in tropical developing countries, especially in the Indian subcontinent [25]. To improve the immune responses to OPV vaccines delivered in large-scale supplementary immunization campaigns, mOPV1 and mOPV3 were introduced in 2005, and bOPV containing types 1 and 3 was licensed and used for the first time in 2009 [35]. The introduction of the mOPV vaccines and bOPV is credited for eliminating the final chains of WPV transmission in India, where the last case of indigenous type 1 wild poliovirus (WPV1) was detected in January 2011, leading to certification of the entire WHO Region of South Asia as free of polio in 2014 by an independent commission . Wild tyDuring the past decade, transmission of remaining WPV1 has been limited to Afghanistan and Pakistan, and the global incidence fell to an all-time nadir of <40 cases per year between 2016 and 2018 . HoweverChallenges not anticipated at time of the World Health Assembly resolution in 1988 have slowed progress and required the commitment of substantial resources to combat, most notably, the emergence and spread of circulating vaccine-derived polioviruses (cVDPVs). By the late 1990s, advances in viral genetic sequencing technology led to the discovery of a circulating type 1 VDPV as the cause of an outbreak of poliomyelitis on Hispaniola, demonstrating for the first time that the attenuated Sabin poliovirus strains in the OPV could mutate, assume the transmissibility and neurovirulence characteristics of WPV, and initiate chains of virus transmission resulting in epidemic transmission . It soonThe withdrawal of Sabin 2, together with the addition to introduction of IPV in the routine immunization schedule, reduced the VAPP burden by about 60%, preventing between 150 and 300 cases annually worldwide. Unfortunately, continuous circulation of cVDPVs that carried over from the preswitch period and the growing type 2 immunity gap that developed after the switch from trivalent OPV to bOPV facilitated more VDPV2 outbreaks than anticipated from contemporary models, and use of mOPV2 vaccine for outbreak control seeded more emergences [39]. Between 2016 and September 2020, cVDPV2 outbreaks spread to 24 countries, mostly in sub-Saharan Africa but also areas of Pakistan, Afghanistan and China, the Philippines, and Malaysia, requiring multiple rounds of mOPV2 or bOPV in response  [40]. InAnother challenge to achieving and maintaining long-term polio eradication is posed by rare persons with inherited immunodeficiency disorders who may excrete VDPV for very long periods and represent a risk to seed new outbreaks where transmission has otherwise ceased . A progrThe challenge of achieving and sustaining eradication has created a clear need for new poliovirus vaccines. New vaccines that build on existing platforms for IPV are being introduced, and considerable progress is being made toward new IPV and new OPV vaccines that are adjuvanted, genetically modified, or rely on new production technologies, to both address current and future needs .As the risk of poliovirus transmission wanes, OPV routine childhood immunization will be replaced country by country with IPV to prevent the emergence of VDPVs. The WHO Strategic Advisory Group of Experts on Immunization has recommended that global use of IPV continue for a minimum of 10 years after OPV cessation and indefinitely in countries that host essential poliovirus facilities, including IPV manufacturers, in order to mitigate the risks of recurrent poliovirus outbreaks seeded by undetected cVDPVs, immune deficient poliovirus excretors, or environmental exposure following accidental release from a manufacturing facility or laboratory [44]. This creates a demand for greater IPV supplies at a cost much lower than the current price in order to make IPV accessible to all low- and moderate-income countries. Various strategies are being developed to meet this need, including antigen sparing and new vaccine production technologies.One practical approach to IPV antigen sparing is use of fractional doses. Two 0.1-mL doses (one-fifth the standard volume) delivered intradermally results in seroconversion rates that are equal to or exceed those from a single full dose administered intramuscularly, while achieving 60% dose sparing [45]. In response to IPV supply constraints, intradermal administration for routine infant immunization has been adopted in India and other South Asian countries. A recent study in Cuba suggests that similar dose sparing can be achieved with fractional doses given intramuscularly, which would abrogate the need for the special equipment and training associated with intradermal administration [46].Antigen sparing can also be achieved by inclusion of an adjuvant, which lowers the antigen contect required to induce an immune response comparable to unadjuvanted IPV. An alum adjuvanted IPV containing one-tenth the antigen content of conventional IPV was recently licensed in Denmark and will soon be available to the global market [47].Escherichia coli, double-mutant labile toxin (dmLT), not only enhances serum neutralizing antibody responses by >5-fold when administered either intradermally or intramuscularly in a murine model, but uniquely induces antipoliovirus immunoglobulin A secretion in the gut, raising the possibility that, unlike conventional IPV, an adjuvanted inactivated vaccine might reduce poliovirus transmission similar to that of OPV . The first-in-human studies with dmLT-IPV are currently in progress.A potent adjuvant derived from Considerable progress has been made toward reduction of long-term risks associated with IPV manufacturing by adoption of seed strains that are less virulent than the wild-type viruses used for conventional IPV production. New Sabin strain vaccines (sIPVs) carry lower biosafety risks in addition to contributing needed supplies to the global market. The sIPV was first licensed in Japan in 2012 in combination with DTP vaccine, and 2 standalone sIPV vaccines have since been approved in China . Additional manufacturers, some in developing countries, plan to produce sIPV for the global market as participants in a WHO-sponsored technology transfer initiative, and recently standardized reagents have been approved by WHO to assist in sIPV development, manufacturing, and regulatory practice [52].Because Sabin vaccine stains still carry a risk of reversion to neurovirulence, evaluation of IPVs produced with “further attenuated” S19 Sabin derivatives that do not replicate in humans is now in progress . In addition, progress has been made in development of IPV with genetically engineered virus-like particles, which would avoid virus cultivation altogether .A project to develop Sabin strain derivatives that are less susceptible to reversion to the wild-type phenotype was initiated by the Bill & Melinda Gates Foundation nearly a decade ago and has now taken on new urgency with the emergence of multiple VDPV2 outbreaks since 2017. Candidate novel oral poliovirus vaccine (nOPV) strains have been generated for all 3 types, with development of nOPV2 given priority. The modifications to the Sabin genome in the nOPV2 vaccine now in clinical development are designed to stabilize key attenuating mutations in the 5′-nontranslated region, suppress recombination with other species C enteroviruses, and limit viral adaptability . Early-pAlthough much progress has been achieved in reducing poliomyelitis incidence and extent of virus transmission since 1988, the eradication target remains elusive and the outlook mixed. Two of the 3 WPV serotypes are certified to be eradicated, and WPV1 transmission appears to have ceased in the WHO African Region, which was recently certified to be free of WPV transmission. The relative rebound of WPV1 cases in Afghanistan and Pakistan since 2018 should not detract from the progress to date, but signals that further intensified efforts will be required to complete interruption of transmission of the only remaining wild-type polioviruses. The success against WPVs must now be balanced against the emergence of cVDPV2 outbreaks in Africa and Asia, which are abetted by use of mOPV2, the only vaccine available to control outbreaks, and the competing global demands imposed by the coronavirus pandemic.Despite the extensive and sustained progress, the GPEI has not achieved eradication. Enormous efforts will be needed to reach the eradication finish line that include new commitment to interrupt WPV1 transmission in Asia and to interrupt transmission of type 2 polioviruses originating from OPVs, in addition to new strategies that include surveillance innovation and development and deployment of new polio vaccines designed to support and sustain the GPEI eradication goals .jiaa622_suppl_Supplementary-MaterialClick here for additional data file.


An improved soil carbon budget serves the dual purpose of promoting soil health, which supports crop productivity, and constituting a pool from which carbon can be converted to recalcitrant forms for long-term storage as a mitigation measure for global warming. In this perspective, we propose the design of crop ideotypes with the dual functionality of being highly productive for the purposes of food, feed, and fuel, while at the same time being able to facilitate higher contribution to soil carbon and improve the below ground ecology. We advocate a holistic approach of the integrated plant-microbe-soil system and suggest that significant improvements in soil carbon storage can be achieved by a three-pronged approach: (1) design plants with an increased root strength to further allocation of carbon belowground; (2) balance the increase in belowground carbon allocation with increased source strength for enhanced photosynthesis and biomass accumulation; and (3) design soil microbial consortia for increased rhizosphere sink strength and plant growth-promoting (PGP) properties.Agricultural cropping systems and pasture comprise one third of the world’s arable land and have the potential to draw down a considerable amount of atmospheric CO From a carbon mass balance perspective, this boils down to increasing soil carbon inputs and storage and decreasing outputs, to attain a net increase in soil carbon storage. We posit that this can be achieved through a comprehensive understanding of carbon fluxes and source-sink interactions in an integrated plant-microbe-soil system. Our rationale for focusing on annual crops are four-fold: (1) according to the U.S. Geological Survey, there is a total of 18.6 million km2 (4.6 billion acres) of cropland globally  as globally .2 at 123 GT year–1 into plant biomass through photosynthesis, of which 3 GT year–1 emanates from anthropogenic activities  emissions  in the top 3 m is 3.3 times the size of the atmospheric pool of 760 GT and includes 1,550 GT of soil organic carbon (SOC) and 950 GT of soil inorganic carbon . The SOCtivities 11, and tectively . Currentmissions . Consequtigation .2) , application of synthetic fertilizers (nitrous oxide), and tillage (CO2) . However2) , up to 1tential” . There atential” . Maybe tSoil organic carbon (as a proxy for soil organic matter) plays two roles as we tackle the challenge of achieving sustainable agroecosystems in the coming decades; by increasing crop productivity and by sequestering atmospheric carbon. SOC promotes crop productivity by improving nutrient retention and water holding capacity, by facilitating efficient drainage and aeration, by minimizing loss of topsoil via erosion, and by providing substrates for the soil microbiomes . SOC canA representation of carbon fluxes in a terrestrial ecosystem is depicted in For carbon, Eq (1) can be summarized as follows:Inputs: Photosynthesis; Soil amendmentsOutputs: Plant and microbial respiration; Emissions of volatile organic compounds (VOCs)Storage: Plant and microbial biomass; Soil carbon as SOC and SIC poolsThe soil carbon content is governed by the balance between photosynthetic inputs via liter and root exudation and amendments such as manure and compost, and outputs through heterotrophic respiration from roots and microbes and via VOC emissions.2 (photosynthate) is shifted between respiration and biomass production, transient and enduring tissues, and aboveground and belowground components. Functional, or optimal equilibrium theory holds that plants allocate resources among organs to optimize whole-plant fitness  of recent photosynthate to their belowground biomass. About 50% of the translocated carbon is used for root growth, while a substantial fraction of this carbon (up to 30%) is further released to the rhizosphere, either as direct root deposition through exudation, sloughed root cap cells, or via mycorrhiza, or is lost through respiration . Both roA substantial portion of the photosynthate can be released to the atmosphere in the form of VOCs, with isoprenoids representing the dominant compound class . The amo2 levels due to climate change, C3 crops that are typically CO2 limited may instead face nitrogen limitation, making a larger root biomass even more advantageous.Soil carbon stocks can be augmented by increasing the rate of carbon additions to the soil, by increasing retention of new carbon deposited in soil, or by reducing the rate of decomposition of SOC already present in the soil. A potential path to increased soil carbon stocks is the employment or development of crop cultivars that input a greater quantity of carbon into the soil through their roots. Just like sink strength of developing grains is a key determinant of grain yield, utilization of carbon for root growth determines root sink strength and belowground accumulation as SOC . This ma2O emissions) might be possible based on specifying feasible increases in total root mass and changing root depth distributions toward those found in perennial grasses. They estimated that widespread adoption of annual crop phenotypes designed to have deeper and larger root systems could yield soil carbon stock increases of 0.5 GT CO2 ha–1 year–1 on current US cropland  quac matter ; (3) chec matter ; and (4)c matter .2. The ideal scenario might be large amounts of high-quality root-derived carbon inputs accompanied by formation of the pore architecture favorable for protecting the new carbon from further decomposition  effects that benefit their host plants, such as conferring enhanced abiotic and biotic stress tolerance and improved nutrient acquisition, including N-fixation . Thus, t2 levels around Rubisco and decreased photorespiratory lost , such as C4 photosynthetic machinery or cyanobacterial carboxysome CCM into C3 crops also hold great potential to dramatically increase source activity  but also by the allocation pattern of photosynthates to other organs, i.e., sink strength. Importantly, if sink strength does not keep up with source strength, this can result in the accumulation of carbohydrates in the leaves and feedback inhibition of photosynthesis . Since erovement . Increas2 through developmental plasticity  slow unnecessary protein turnover; (2) replacing, relocating, and/or rescheduling metabolic activities; (3) suppressing futile cycles; (4) improving ion transport efficiency (13C) and nitrogen (15N) fluxes within a model cereal could be instructive, displaying the proportion of carbon allocated to the source and sinks (grain and roots), and the percentage released to the soil and the atmosphere. This integrated understanding of carbon movement would be viewed in relation to plant growth, biomass, yield, and plant productivity. This data would serve as a blueprint from which to understand developmental and environmental shifts in carbon allocation, to begin engineering strategies, and to better support predictive models. For example, toward understanding the effects of reduced nitrogen, higher CO2, drought, and heat, singly, and in different combinations, on the plant-microbe-environment carbon continuum. Another example to highlight is the need for measuring and predicting fluxes relates to the emissions of N2O (the most potent biogenic greenhouse gas on a per mass basis) resulting from increased nitrogen inputs when needed to match elevated atmospheric CO2 levels. Models are also required that account for sink stimulation of photosynthesis to assess the extent by which carbon costs incurred as part of interactions or symbioses with the rhizosphere microbiome are compensated for by enhanced photosynthesis and biomass production  increased belowground carbon allocation for larger and deeper root biomass; (2) interactions with a tailored, synthetic soil microbiome for increased rhizosphere sink strength and enhanced PGP properties that facilitate nutrient acquisition and water-use efficiency; and (3) increased source strength for enhanced photosynthesis and biomass accumulation. This represents an ambitious initiative that entails genome engineering/editing of the integrated plant–microbe–soil system, supported by systems-level multi-omics analysis, and metabolic flux analysis and modeling. It will also be important to engage international breeding programs and cull resources from extensive germplasm collections (Carbon farming aims to improve the rate at which COlections . This beWe conclude by recognizing that soil carbon storage is not an infinite solution to curtailing greenhouse gas emissions, primarily since soils have an upper limit or saturation level of carbon . We recoThe original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.CJ conceived the work. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ferroptosis is a novel form of programmed cell death, and it is characterized by iron-dependent oxidative damage, lipid peroxidation and reactive oxygen species accumulation. Notable studies have revealed that ferroptosis plays vital roles in tumor occurrence and that abundant ferroptosis in cells can inhibit tumor progression. Recently, some noncoding RNAs (ncRNAs), particularly microRNAs, long noncoding RNAs, and circular RNAs, have been shown to be involved in biological processes of ferroptosis, thus affecting cancer growth. However, the definite regulatory mechanism of this phenomenon is still unclear. To clarify this issue, increasing studies have focused on the regulatory roles of ncRNAs in the initiation and development of ferroptosis and the role of ferroptosis in progression of various cancers, such as lung, liver, and breast cancers. In this review, we systematically summarized the relationship between ferroptosis-associated ncRNAs and cancer progression. Moreover, additional evidence is needed to identify the role of ferroptosis-related ncRNAs in cancer progression. This review will help us to understand the roles of ncRNAs in ferroptosis and cancer progression and may provide new ideas for exploring novel diagnostic and therapeutic biomarkers for cancer in the future. Programmed cell death (PCD) is important for the balance between the progression of diseases and human health Ferroptosis is regulated by specific signal transduction pathways through iron accumulation, lipid peroxidation and cellular membrane destruction, and ferroptosis can be modulated by drugs or genetic interventions Iron accumulation plays a critical role in producing ROS via the Fenton reaction and enzyme activity in terms of lipid peroxidation. Although iron is essential in physiological processes, excessive iron is pernicious and can trigger ferroptosis. Ferroptosis is strictly regulated by modulators related to iron metabolism processes, such as iron intake, stockpile, usage, and release - or glutathione peroxidase 4 (GPX4) leads to decreased production of reduced GSH 2) antiporter of system Xc- is composed of solute carrier family 3 membrane 2 (SLC3A2) and solute carrier family 7 membrane 11 (SLC7A11) and can import extracellular Cys2 into cells and exchange intracellular Glu. Moveover, some evidence has revealed that the cargo receptor SQSTM1/p62 can induce the autophagic degradation of ARNTL, known as clockophagy, and promote lipid peroxidation and ferroptosis via the EGLN2/HIF1A pathway Lipid peroxidation leading to cell membrane destruction is the central intermediate link of ferroptosis. The molecular mechanism of lipid peroxidation is that the inhibition of system Xc-, which involves GSH, is one of the main antioxidant defenses in our body. When system Xc- is inhibited by erastin, Cys2 cannot influx cells, resulting in a decrease in the amount of Cys2. Due to the important role of Cys2 in the process of GSH biosynthesis, Cys2 deficiency can reduce the level of GSH. Then, the exhaustion of GSH can reduce the expression and activity of GPX4 - is one of the main biochemical features of ferroptosis, and the regulation of ferroptosis is the primary way to control its occurrence.System Xc- via certain compounds, such as erastin, sulfasalazine -. These genes regulate the transcription or translation of subunits of system Xc-, such as SLC7A11 or SLC3A2, affecting the biological processes of the ferroptosis. The tumor suppressor BRCA1-related protein1 (BAP1) could inhibit the SLC7A11 expression and then lead to elevated lipid peroxidation and ferroptosis 2, otherwise NF E2-related factor 2 (NRF2) plays the opposite role in SLC7A11 6A reader identified in 2017, suppresses the expression of SLC3A2 by inhibiting HOXA13, a transcription factor of SLC3A2 expression, to trigger system Xc- -dependent ferroptosis Several studies uncovered that ferroptosis could be induced by inhibiting system XcGPX4, another main antioxidant defense, can directly reduce cell membrane phospholipid hydroperoxide to hydroxyphospholipid, taking advantage of GSH as a substrate, resulting in the suppression of ferroptosis in cancer cells Genetic depletion of GPX4 causes lipid peroxidation and then induces ferroptosis in cancer cells or tissues 2+ and lipid peroxidation. When the two antioxidant defenses, GSH and GPX4 systems, are impaired, the accumulation of toxic ROS will occur and then induce cell death. Some studies have revealed that erastin causes the production of ROS in some cell lines In the process of lipid peroxidation, the lethal accumulation of lipid ROS can destroy the cell membrane, leading to ferroptosis -, and then inhibit cancer growth Many studies have indicated that ferroptosis plays a crucial role in the regulation of the pathological process of cancer Ferroptosis is related to the prognosis of many types of cancer, but we know little of the mechanism of the ferroptosis in cancer, especially regarding the role of ferroptosis-related ncRNAs in cancer. Ferroptosis is tightly related to noncoding RNAs (ncRNAs) and cancer 2+In terms of regulating ferroptosis-related genes, some studies have demonstrated that many ncRNAs play vital roles in regulating the expression of the ferroptosis-related genes - and GPX4, and several ncRNAs play important roles in the process of lipid metabolism. Many ncRNAs regulated the protein level of SLC7A11 Regarding lipid peroxidation, ncRNAs regulate the subunits of system XcRecently, an increasing amount of evidence has demonstrated that ncRNAs play an important regulatory role in cancer progression via the ferroptosis pathway Lung cancer is the leading cause of cancer-associated deaths, with approximately 1.8 million deaths (18%), and it is the most common cancer in men worldwide, with an estimated 1.44 million new cases each year (14.3%) Recently, miRNAs have attracted substantial attention in lung cancer. Several miRNAs play important roles in chemotherapeutic resistance via ferroptosis. For example, exosomal miR-4443 could increase cisplatin resistance to reduce the therapeutic effect of NSCLC via METTL3/FSP1-induced ferroptosis Furthermore, lncRNAs also play relevant roles in lung cancer progression. In 2018, Chao Mao However, more evidence is needed to explore the regulatory mechanism of ncRNAs in lung cancer via the ferroptosis pathway, and the regulatory roles of other ncRNAs in ferroptosis in lung cancers remain to be discovered.Gastrointestinal cancer is also a serious leading cause threatening the health of humans worldwide, and its death rate is estimated to be 1.69 million (17.1%) To explore the molecular mechanism of UGC, Ahmed Gomma Not only do miRNAs play regulatory roles in the proliferation and metastasis of gastrointestinal cancer, but lncRNAs also have important effects on improving gastrointestinal cancer progression In addition to gastrointestinal cancer, liver cancer is the third largest cause of cancer-triggered death, with approximately 8.3% in both sexes 2+, and reduced the GSH levels when HepG2 and Hep3B cells were treated with erastin, whereas overexpression of anti-miR-214 demonstrated the opposite effect. In addition, some studies began to focus on the role of lncRNAs in the regulation of cellular ferroptosis in HCC. In 2019, Wenchuan Qi et al. Several studies have focused on ferroptosis in liver cancer and explored its molecular mechanism in depth in recent years. Some studies have revealed the role of ncRNAs in the regulation of ferroptosis in HCC Similar to the roles of miRNAs and lncRNAs in liver cancer, circRNAs also demonstrate crucial regulatory roles in liver cancer cell ferroptosis. One study revealed that circIL4R, which is greatly upregulated in HCC tissues and cells, could inhibit ferroptosis by sponging miR-541-3p through the GPX4 pathway, resulting in the promotion of HCC tumorigenesis Overall, ncRNAs may become potential therapeutic targets to treat HCC via the ferroptosis pathway.Breast cancer has exceeded lung cancer as the most frequently newly diagnosed cancer in both sexes and has the most cancer-related deaths among women worldwide. There were an estimated 2.3 million new cases (11.7%) in both sexes and 0.68 million deaths (15.5%) in females To improve the therapeutic effects of drugs, several studies have revealed that some drugs exhibit anticancer effects by regulating ncRNA expression to affect cancer cell ferroptosis Urogenital cancer is also one of most common cancers, but its newly diagnosed cases and deaths are lower than those of female breast cancer. It consists of bladder cancer, renal cancer, ureteropelvic cancer and urinary tract cancer. Among these cancers, bladder cancer and renal cancer accounted for an estimated 1 million new cases (5.5%) and 0.37 million deaths (3.9%) worldwide To explore novel diagnostic and therapeutic targets, accumulating evidence has focused on the role of ferroptosis-related ncRNAs in the genesis, progression, and treatment of urogenital cancer The number of new cases of prostate cancer is very high, next only to lung cancer in males, at an estimated 14.1%, but the cancer-related death rate is lower (approximately 4.5%) than that of lung cancer (approximately 21.5%) Cervical cancer is the fourth most common cancer in women, with an estimated 6.5%, and ranks as the fourth leading cause of cancer-associated death in women, with approximately 7.7% Ovarian cancer is also a common gynecological cancer, with its number of deaths ranking eighth among gynecological cancers Acute leukemia (AL), including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), is also a serious malignant disease However, ncRNAs, including miRNAs, lncRNAs and circRNAs, require further investigation regarding the chemotherapeutic resistance of AML.Although less common in other cancers, glioma is the most common primary central nervous system tumor and accounts for approximately half of all primary intracranial tumors. The five-year survival rate of adult high-grade glioma is very low. According to the WHO grade, low-grade glioma is grade I or II, and high-grade glioma is grade III or IV among the four grades of glioma Head and neck squamous cell carcinoma (HNSCC) is a series of malignant tumors involving many tissues in the head and neck region, including the oral cavity, nasopharynx and throat In contrast to other cancers, melanoma stemming from melanocytes is not common. However, melanoma is the third most frequently occurring malignant tumor of the skin. Melanoma lacks specific treatment, except for surgical resection in the early stage. Hence, ferroptosis is a focus of researchers, and new cell death contributes to inhibiting melanoma progression. Meiying Luo 2+ content in clinically relevant radioresistant (CRR) cells. Oxidative stress and ferroptosis in CRR cells are inhibited. In the future, more investigations will be required to uncover the mechanism by which miRNAs improve radioresistance in cancer cells via the ferroptosis pathway.Like chemotherapy, radiation therapy (RT) is also one of the most common therapies for cancers. However, radioresistance decreases the therapeutic effect of RT, and the mechanism of radioresistance is not well understood. In recent years, Kazuo Tomita Ferroptosis, a newly discovered form of programmed cell death, is related to some pathophysiological processes, especially many types of cancers. Numerous studies have focused on exploring the regulatory mechanisms of cancers via the ferroptosis pathway. Great progress in exploring the regulatory role of ncRNAs in cancers by ferroptosis has been made. Taken together, these findings contribute to further understanding the pathogenesis of cancers and have demonstrated that ferroptosis-associated ncRNAs may act as a series of tumor inhibitors to suppress cancer growth. In addition, ncRNAs, including miRNAs, lncRNAs and circRNAs, have the potential to be novel anticancer therapeutic methods and diagnostic biomarkers by regulating the ferroptosis of cancer cells Table .Many studies have revealed that these ncRNAs play important roles in the progression of cancers via ferroptosis and that these ncRNAs may regulate the ferroptosis of cancer cells to induce or inhibit tumorigenesis. It has been demonstrated that lncRNAs and circRNAs always sponge miRNAs to regulate the expression of GPX4 and induce lipid peroxidation of the cellular membrane. Furthermore, abnormal lipid peroxidation destroys cancer cell membranes, resulting in the ferroptosis of cancer cells. Sometimes, these ncRNAs can directly target GPX4, SLC3A2 and SLC7A11 to induce ferroptosis in cancer cells. In addition, some ncRNAs regulate the ferroptosis of cancer cells via the lethal accumulation of ROS and the abnormal metabolism of iron.Due to the individual heterogeneity of ncRNA expression in different types of cancer, ncRNA-associated therapy and biomarkers may be applied to support personalized cancer treatment. Although an increasing number of studies have revealed a regulatory mechanism between cancer and ferroptosis, a deeper understanding of the mechanism by which ferroptosis-related ncRNAs regulate the progression and growth of cancer is still needed. Moreover, future studies should pay more attention to the role of ncRNAs in the linkage between cancer and ferroptosis. Moreover, some biomaterials, like nanomaterials, may overcome the shortcomings of conventional therapeutic schedules for tumor-targeted ferroptosis therapy by preloading antitumor drugs This review has summarized the regulatory roles of several types of ncRNAs in cancer progression and ferroptosis. These studies are beneficial for understanding the pathogenesis of cancer. Ferroptosis-related ncRNAs have great potential to act as anticancer therapeutic targets by regulating ferroptosis. Targeting these key ncRNAs may reveal novel therapeutic methods or diagnostic biomarkers to inhibit the growth and progression of malignant tumors.


We report a disease and mortality event involving swans, seals, and a fox at a wildlife rehabilitation center in the United Kingdom during late 2020. Five swans had onset of highly pathogenic avian influenza virus infection while in captivity. Subsequently, 5 seals and a fox died (or were euthanized) after onset of clinical disease. Avian-origin influenza A virus subtype H5N8 was retrospectively determined as the cause of disease. Infection in the seals manifested as seizures, and immunohistochemical and molecular testing on postmortem samples detected a neurologic distribution of viral products. The fox died overnight after sudden onset of inappetence, and postmortem tissues revealed neurologic and respiratory distribution of viral products. Live virus was isolated from the swans, seals, and the fox, and a single genetic change was detected as a potential adaptive mutation in the mammalian-derived viral sequences. No human influenza-like illness was reported in the weeks after the event.  Phoca vitulina), 1 juvenile gray seal , and 1 juvenile red fox (Vulpes vulpes) died or were euthanized over a 2-day period. The fox died suddenly after a short period of nonspecific malaise and inappetence. The seals exhibited sudden-onset neurologic signs, including seizures before death or euthanasia  held in isolation at the center because of acute-onset malaise and terminal seizures. The 5 swans were submitted for examination and testing under the Avian Influenza Wild Bird Surveillance Scheme   full postmortem examination (PME). Two swans were submitted frozen and did not undergo PME, but we took oral and cloacal swab samples from these carcasses for virologic testing. We examined plain swab samples from the oropharynx and cloaca of each of the swans, and similarly from the nasal cavity and rectum of each of the 3 seals, for preliminary influenza virologic testing. After receipt of the avian influenza virus results, we sampled the 3 freshest seal carcasses because they were more likely to yield meaningful results and have suitable samples for histopathologic examination. We took tissue samples of brain, liver, kidney, spleen, lymph nodes, and lung from the seals and the fox and stored them at −80°C until required.https://en.ssi.dk), as previously described (https://www.bio-rad.com) in selected fox tissues.For histologic examination, we took samples of heart, lung, liver, spleen, and brain tissues from the swans, fox, and seals, as well as lymph nodes of the mammalian species, and fixed them in 10% neutral buffered formalin before processing for hematoxylin and eosin staining and immunohistochemical (IHC) examination. We performed IHC examination by using anti–influenza A nucleoprotein primary antibody  assay (https://www.illumina.com) (https://platform.gisaid.org). We deposited sequences in the GISAID database .We performed virus isolation on PCR-positive samples from the swans, seals, and fox by using specified pathogen-free embryonated fowls’ eggs . This facility was designed to minimize transmission of microorganisms among residents; animals in separate cubicles had no direct contact. Basic biosecurity practices were in place, such as decontamination steps between cubicles and staff using respiratory protective equipment  and dedicated personal protective equipment for each cubicle.Anas platyrhynchos) housed across 3 rooms in groups of 10. No clinical disease or deaths were recorded in these birds.The 5 juvenile mute swans were rescued from different locations and brought to the center for treatment during October 23–November 20, 2020 . These bThe 4 common seals were estimated to be 5–6 months of age and arrived individually at the facility 1–2 months before the disease episode , admitteThe gray seal was a 2-week-old pup admitted for care after maternal abandonment 2 weeks earlier . It was The fox had been in the isolation unit of the center for 2 weeks  in cubicThe body condition of the 3 swans examined ranged from good to poor. Gross findings among the carcasses included petechiae in the liver and epicardium in 1 bird and opacity of the air sacs in another. Microscopic examination of tissues from 1 bird revealed multifocal, necrotizing, nonsuppurative myocarditis, hepatitis, splenitis, nephritis, and encephalitis, along with intralesional presence of influenza A virus antigen, observed during IHC examination.The body condition of the 3 examined seals was judged to be fair. Gross examination of 2 common seals revealed generalized lymphadenomegaly and multiple pale foci in the lungs. One common seal also showed congested meninges. The gray seal pup showed generalized lymphadenomegaly but no other gross changes. Microscopic examination of the seal tissues revealed mild, eosinophilic, interstitial pneumonia in the 2 common seals, consistent with lungworm infection, and severe, necrotizing, nonsuppurative polioencephalitis in the 2 common seals and the gray seal. The lymph node sections showed nonspecific reactive hyperplasia, accounting for the gross enlargement of these organs. IHC examination for influenza A virus revealed multifocal immunolabelling in the neurons within the gray matter of the brain in all 3 seals, in close association with the inflammatory lesions , panel AThe body condition of the fox was judged to be poor. The animal was visibly underweight, and gross examination revealed large areas of alopecia and crusts affecting the skin of the body and limbs, mild splenomegaly, and generalized reddening of the lungs. Microscopic examination of the brain, lung, and heart revealed severe, acute, nonsuppurative, polioencephalitis , panel BOropharyngeal and cloacal swabs from all 5 swans tested positive for H5N8 (Appendix Table). WGS data were generated for 1 of the swans submitted (A/mute swan/England/234135/H5N8 2020/12/01).Nasal and rectal swab samples taken during PME of the 3 seals tested negative by PCR for influenza A virus. However, we retrospectively detected influenza A(H5N8) virus nucleic acid in the brain and lung of the gray seal and the brain of 2 common seals (Appendix Table). We performed virus isolation on pooled samples and successfully isolated virus from the pooled brain tissue of seals .We detected influenza A virus RNA in each of the fox samples , and PCR subtyping determined the influenza A virus subtype as H5N8 (Appendix Table). We isolated virus and generated WGS from the fox brain tissue (A/red fox/England/AVP-M1-21-01/2020 (H5N8)). The hemagglutinin cleavage site (CS) motif from selected fox and seal samples had the amino acid sequence PLREKRRKRGLF, consistent with 99% of CSs characterized across highly pathogenic avian influenza (HPAI) H5N8 virus sequences from the United Kingdom during autumn and winter 2020–2021. Analysis of WGS data generated from the fox, seal, and swan samples demonstrated high sequence similarity . Alignment of the viral hemagglutinin  and neuraminidase genes  demonstrated that virus from the fox and seals clustered closely with the viruses detected in the swans from the same wildlife center. Comparison of WGS data from the fox, seal, and swan identified a total of 33 aa substitutions that have been associated with altered virulence reported through natural or experimental infections. Almost all substitutions have been identified in the H5N8 virus sequences generated during the 2020–2021 epizootic in avian species . The genomes of the fox, seal, and swan-derived viruses were homologous at the amino-acid level, with the exception of a single amino-acid substitution at position 701 in the polymerase basic protein 2 gene (D701N) that was only present in the fox and seal sequences. Analysis of WGS data obtained from the fox samples demonstrated a lack of other viral agents, including canine distemper virus.At the time of the outbreak, the rehabilitation center fell within a government-mandated protection zone because of previously confirmed detection of HPAI virus infections; consequently, moving birds off the center was restricted around the time of the disease episode. Restrictions were also applied specifically to the rehabilitation center when suspicion of notifiable disease in the swans was reported. The cases investigated in this outbreak were limited to animals held within the quarantine ward, and over the subsequent months, no further cases of unusual or unexplained clinical disease or death occurred among the mammals and birds at the center. After the disease event, but before diagnosis of H5N8 in the seals and fox, the center was visited as part of surveillance activities within the protection zone. Resident bird species were examined for evidence of clinical disease, and 38 birds  were sampled for virologic testing by PCR. All birds tested negative. Blood was not taken for analysis.We determined that avian-origin influenza A(H5N8) virus was the cause of death in a red fox and the cause of seizures in a gray seal and several common seals housed at a wildlife rehabilitation center. These events occurred roughly 1 week after 5 swans housed in the same quarantine unit died from infections with HPAI H5N8 virus. Genetic and epidemiologic investigations suggest that the swans were most likely the source of infection for the fox and seals; virus transmission likely occurred by fomite transfer or aerosol spread.The severity of disease and pathologic findings in the seals and fox was unexpected. High-mortality outbreaks of disease associated with influenza A virus have been described in common seals and were associated with virus subtypes H10N7  virus caused severe disease and death in juvenile seals and a fox held in a wildlife rehabilitation center, in addition to swans that had also succumbed to the virus. All evidence suggests that the swans were the most likely source of infection for the fox and seals. The location of affected animals within the quarantine facility  suggests either aerosol or fomite spread as the likely cause of dissemination of infectious virus between cubicles. Although the quarantine facility is designed to limit spread of infectious microorganisms through the use of good basic hygiene practices, it is not a biosecure facility designed to handle Biosafety Level 3 pathogens; as such, highly transmissible agents such as avian influenza may well spread even with some infection prevention measures in place. Because influenza infection was not suspected at the time of the event, biosecurity practices at the center may have been less effective at preventing spread compared with those implemented at a heightened level of biosecurity, which would likely have been in place had there been an awareness of the presence of influenza infection. Determining the cause of disease in the seals and fox retrospectively was entirely reliant on collaboration between field veterinary services, pathologists, and virologists, and this case highlights the importance of wildlife disease surveillance. Although genetic analyses indicated no increased risk for human infection with the H5N8 viruses in this outbreak, the investigation shows how these viruses may have unexpected and severe health risks for mammalian species. Such spillover disease events in atypical host species constitute additional factors for veterinary authorities to consider during disease outbreaks and highlight the importance of wildlife disease surveillance that uses interdisciplinary and collaborative approaches.Additional information about encephalitis and death in wild mammals at a rehabilitation center after systemic infection with highly pathogenic avian influenza A(H5N8), United Kingdom.


Lipofuscin is an intracellular aging pigment with fluorescent properties, found in retinal pigment epithelium cells of the eye. It is the main fluorophore used in fundus autofluorescence imaging techniques to diagnose, describe, and follow retinal disease. Lipofuscin forms by incomplete lysosomal degradation of cellular material previously subjected to oxidative changes. A2E is the most studied fluorescent component of lipofuscin, but most of its composition remains unknown. Lipofuscin is photoreactive, generating reactive oxygen species, which may explain its role in disease development. Further knowledge is needed concerning lipofuscin genesis, biochemical composition, fluorescent compounds, and role in pathogenesis of retinal degenerative disease. The technique uses fluorescent properties of the natural fluorophore lipofuscin, an aging pigment, that progressively accumulates within RPE cells. A2E has long been considered one of the main components of lipofuscin with fluorescent properties and a noteworthy role in retinal degenerative disease. Recent knowledge about A2E has shown that this compound, although important, does not completely explain the fluorescent and pathologic properties of lipofuscin . It is a ubiquitous yellow-brown aging pigment that is stored particularly within postmitotic cells with high metabolic activity. This includes cardiac myocytes, neurons and RPE. Lipofuscin is heterogenous in origin, composition, and biochemical qualities, depending on the cell type in which it accumulates. Lipofuscin is the name given to nondegradable aggregates consisting of lipids and proteins that progressively accumulate throughout life within the lysosomal compartment of various cells of the body [5].We believe it is important to concisely mention ceroid, an intracellular pigment like lipofuscin. Unlike the latter, ceroid is strictly a pathological pigment and is present in a series of central nervous system degenerative disease called neuronal ceroid lipofuscinoses [Lipofuscin and retinal pigment epithelium4]. These granules are membrane-bound and lay in the basal half of RPE cells. They measure between 1 to 2 μm in diameter, with undulated or scalloped outlines [6]. In aged individuals, lipofuscin granules may conjugate with melanin forming aggregates called melanolipofuscin [7]. Lipofuscin and melanolipofuscin granules may occupy up to one third of the cytoplasmic space of RPE cells in persons aged over 70 [8]. Lipofuscin forms clusters of granules inside the RPE [9]. The topographic distribution of lipofuscin across the retina appears to follow that of rod cells . Lipofuscin content of RPE increases with age until about 70 years and then plateaus or decreases  with a concomitant decrease in melanosomes and increase in melanolipofuscin granules [10]. These spectral properties allow for noninvasive imaging of lipofuscin content of the RPE.Lipofuscin granules inside RPE cells demonstrate a characteristic orange-red or golden-yellow autofluorescence when excited with ultraviolet or blue light . A significant amount of RPE lipofuscin may in fact be due to autophagy as in vitro studies have shown that lipofuscin-like granules are present in RPE cells even in the absence of phagocytic challenge of photoreceptor outer segment [13].Lipofuscin may be formed by two ways: either by autophagy  or by phagocytosis of other cellular components, as in the case of the RPE, which functions to phagocytose photoreceptor external segments [Biochemical composition of lipofuscin9]. The photoreceptor/ RPE complex is considered highly susceptible to oxidative modification due to its important exposure to sunlight, elevated oxygen levels in circulating blood and high content of polyunsaturated fatty acids (PUFAs) in mitochondrial and cell membranes . These oxidized products are further modified within lysosomes [9]. Evidence that these alterations take place is the fact that the spectral properties of lipofuscin fluorophores differ from those of their oxidized precursors [15].The biochemical composition of lipofuscin is complex, comprising variable amounts of lipids, proteins, carbohydrates and metal ions, its composition depending on the tissue of origin. Incomplete degradation of cellular material within the lysosomal compartment is due to oxidative changes of these compounds either by normal cell metabolism or oxidative stress [15]. These aldehydes represent the precursors of fluorescent products. Malondialdehyde (MDA) and 4-hydroxynonenal (HNE) have been most extensively studied. MDA further reacts with amino acids in low-pH conditions to form Schiff bases with aminopropane structure (-N=CH-CH=CH-NH-). These Schiff bases have emission in the 450-470 nm region when excited by ultraviolet light. This could explain some of the fluorescent properties of lipofuscin. Schiff bases are also created by interaction between reducing sugars and amino compounds, a process called nonenzymatic glycosylation or Maillard reaction. Schiff bases are then converted to ketoamines, called Amadori products, which are further degraded to unsaturated carbonyls, the analogues of lipid peroxidation products. Although the list of age-pigment like fluorophores is more extensive than our description , all appear to form through a common conjugation between amino compounds and carbonyls.Reactive oxygen species from cell metabolism or oxidative stress induce lipid peroxidation, resulting in lipid hydroperoxides or cyclic peroxides, which further degrade to form carbonyl compounds, mainly aldehydes [17] have shown that that purified lipofuscin granules from RPE cells contain only small amounts of protein and that modified lipids from outer segment and mitochondrial membranes are the major contributors, both to lipofuscin composition and to its presumed phototoxic activity. Despite the numerous attempts over the last decades to describe the complex composition of lipofuscin in RPE cells, its constituents remain mostly unknown. A proteomic study done by Ng et al. [A2E – A key component of lipofuscin?18] separated lipofuscin from donor eyes into 3 phases using methanol and chloroform: a polar (methanol-water) phase, a nonpolar (chloroform) phase and an insoluble fraction. The polar phase emitted only a faint fluorescence and was not further characterized. Using thin layer chromatography, they analyzed the nonpolar fraction and identified 10 fluorophores with different emission spectra. It is important to mention that not all the age-related fluorescent material was extracted and that the fluorescent insoluble fraction increased with age. To characterize the chromatographic and spectral properties of the fluorescent components of human RPE lipofuscin, Eldred and Katz [19] showed that the main orange-emitting fluorophore is N-retinylidene-N-retinylethanolamine, a pyridinium bis-retinoid synthetized using 2 molecules of all-trans-retinal and 1 molecule of ethanolamine (A2E). A2E has an amphiphilic structure responsible for its detergent-like action in membrane disruption. Its quaternary amine structure also inhibits lysosomal action by binding to certain lysosomal enzymes [19]. In addition to A2E, lipofuscin contains molecules structurally related to A2E resulting from autooxidation or photooxidation of A2E [20]. The composition of the insoluble part of lipofuscin remains unknown. This fraction may also play a significant role in photoreactivity of lipofuscin granules [21].Later, Eldred and Lasky [Spectral characteristics of lipofuscin22] studied the spectral characteristics of lipofuscin granules and melanosomes in human RPE cells with respect to donor age. When excited at 364 nm, lipofuscin granules showed a broad emission spectrum with a peak at 600 nm. The same emission peak was observed when an excitation of 476 nm was employed, a value like the excitation employed by in vivo autofluorescence systems [9]. When comparing emission spectra from different age groups, three other spectral regions were of interest: a blue-green shoulder at 470 nm and a green-yellow shoulder at 550 nm were evident in 4–29-year-old and 30–49-year-old but absent in > 50-year-old. A third red shoulder at 680 nm was consistent in the > 50-year-old group. Emission in blue decreased with increasing age while emission in red increased. The fluorescence intensity of lipofuscin granules increased with increasing age. Both the emission and the excitation spectra peaks were 40% higher in older individuals when compared with younger ones. Melanolipofuscin has spectral characteristics intermediate between those of lipofuscin and melanosomes [9].Boulton et al. [Lipofuscin phototoxicity23]. The photooxidative damage appears to be greater in aged RPE because of a greater number of lipofuscin granules as well as an overall increased photoreactivity of lipofuscin with increasing age [21]. In conditions simulating in vivo conditions of human RPE cells (light exposure and aerobic conditions relating to high oxygen tension of circulating blood), RPE lipofuscin shows notable photoreactivity. Lipofuscin has phototoxic potential that appears to be wavelength-dependent: exposure of lipofuscin-containing cells to short-wavelength visible light (390-550 nm) caused lipid peroxidation, with increased levels of MDA and HNE, protein oxidation with formation of protein carbonyl, loss of lysosomal integrity, cytoplasmic vacuolation and membrane blebbing. The cytologic damage culminated in cell death. Exposure to wavelengths of 550 to 800 nm did not show any adverse effects . Lipofuscin generates reactive oxygen species (ROS), including singlet oxygen (1O2), superoxide anion (O2-) and hydrogen peroxide (H2O2), as well as lipid hydroperoxides and MDA, which are suggestive of lipid peroxidation. Photoreactivity is also wavelength-dependent [9], its accumulation in RPE cells concurring with the development of AMD. Furthermore, retinal areas with high amounts of lipofuscin seem the most susceptible to degeneration [26]. The most studied component and potential phototoxic component of lipofuscin is A2E. The evidence has shown that A2E has weak photoreactivity and its contribution to RPE cytotoxicity is arguable [24]. Another source of photoreactivity may be represented be A2E-derived molecules, such as the epoxide nonaoxirane [27]. The insoluble fractions of lipofuscin may also represent an important source of photoreactivity .Because of its photoreactivity, lipofuscin may contribute to the pathogenesis of age-related macular degeneration (AMD) . Across various retinal degenerative disease, it is still difficult to determine if lipofuscin is the causative agent of disease or its consequence. In the case of AMD, there is significant evidence of the pathogenic role of lipofuscin in this disease, either by exhibiting oxidative stress and chronic dysfunction of RPE cells  or by its inflammatory  and angiogenic component [32].Autofluorescence imaging of the retina shows elevated levels of lipofuscin associated with retinal degeneration [Fundus autofluorescence22]. Initially, retinal autofluorescence was assessed by fundus spectrofluorometers, designed to measure excitation and emission spectra from small retinal areas, only 2 degrees in diameter. Fundus fluorescence revealed a maximum emission at 630 nm and peak excitation at 510 nm. It also showed topographic variability across the retinal fundus, with a foveal minimum, a maximum at 7° to 15° from the fovea and a decrease towards the periphery [33]. This is determined by a lower amount of lipofuscin in the foveal RPE cells and absorption of the excitation light by macular pigment [9].As mentioned earlier, lipofuscin in the retina shows a characteristic autofluorescent pattern . Currently commercially available cSLO systems used to obtain fundus autofluorescence images are the HRA Spectralis platform  and the Nidek F-10 Digital Ophthalmoscope . Spectralis HRA+OCT also allows simultaneous cSLO and optical coherence tomography (OCT) recordings [9]. The cSLO system is based on excitation of a blue laser, with a wavelength of 488 nm and recording of emitted light above 500 nm.Fundus autofluorescence imaging has gone a long way since the use of spectrofluorometers and current imaging methods use confocal scanning laser (cSLO) systems and modified fundus cameras, with ultra-wide-field imaging and different excitation wavelengths to evaluate retinal autofluorescence [Fundus autofluorescence in ophthalmology36-40].Fundus autofluorescence allows mapping of in vivo lipofuscin distribution across the retinal fundus. This technique is fast, easily repeatable, and noninvasive. Unlike other imaging techniques (fluorescein angiography or indocyanine green angiography) it does not require the use of a fluorescent dye. It is currently regarded as having an important contribution in multimodal imaging, as a complementary imaging technique to fundus photography, angiography, and OCT in the diagnosis of various retinal disease, in determining disease prognosis and evolution over time and in monitoring disease response to treatment [For example, fundus autofluorescence has been used to monitor the idiopathic macular hole.In eyes with macular hole, the lack of lutein and zeaxanthin pigments in the fovea results in hyperautofluorescence, although hypoautofluorescence is normally noted at the fovea.Despite our knowledge regarding lipofuscin composition, its interaction with light and its association with retinal degenerative disease, many questions still exist. We must still determine the full spectrum of fluorescent compounds of lipofuscin. We know that A2E is one of them but we also know that its contribution to lipofuscin fluorescence is small. Further characterization of lipofuscin composition, including spectral analysis, may improve current imaging techniques or enhance development of novel ones.A similar question is asked when we consider the role of A2E in retinal degenerative disease: what component of lipofuscin is pathogenic? An even more complex question is if lipofuscin is at all a cause of retinal degenerative disease or just its consequence. And if progressive accumulation of lipofuscin undeniably leads to retinal damage, is there a way to prevent its formation? We also have very limited information about melanolipofuscin granules, their potential usefulness in detecting retinal disease and/ or understanding the pathophysiology of certain disease.Conflict of Interest statementAuthors state no conflict of interest.AcknowledgmentsAll the authors had equal contributions. Sources of FundingNo funding was received.DisclosuresNil.Authors’ contributionsOM and EFC designed the study and are responsible for the acquisition and interpretation of the data.SIP and CCC were involved in the design of the study, inspecting carefully, and revised the manuscript.CP was involved in the conception and drafting of the study and revised the manuscript. All authors read and approved the final manuscript.Authors’ informationThe author Stella Ioana Popescu (Patoni) is a PhD student in the Ophthalmology Department of “Victor Babeş” University of Medicine and Pharmacy in Timișoara, Romania, but she does not work either in the Ophthalmology Department or the faculty. Her affiliation must be this university to have her article validated for her doctoral thesis. Her affiliation with the Ophthalmology Department is because she is a PhD student.


To date, nearly 100 autoimmune diseases have been an area of focus, and these diseases bring health challenges to approximately 5% of the population worldwide. As a type of disease caused by tolerance breakdown, both environmental and genetic risk factors contribute to autoimmune disease development. However, in most cases, there are still gaps in our understanding of disease pathogenesis, diagnosis, and treatment. Therefore, more detailed knowledge of disease pathogenesis and potential therapies is indispensable. DNA methylation, which does not affect the DNA sequence, is one of the key epigenetic silencing mechanisms and has been indicated to play a key role in gene expression regulation and to participate in the development of certain autoimmune diseases. Potential epigenetic regulation via DNA methylation has garnered more attention as a disease biomarker in recent years. In this review, we clarify the basic function and distribution of DNA methylation, evaluate its effects on gene expression and discuss related key enzymes. In addition, we summarize recent aberrant DNA methylation modifications identified in the most important cell types related to several autoimmune diseases and then provide potential directions for better diagnosing and monitoring disease progression driven by epigenetic control, which may broaden our understanding and contribute to further epigenetic research in autoimmune diseases. Autoimmune diseases, which represent a family of almost 100 conditions, have received mounting and widespread attention due to their complex etiologies and the life-long threat they pose. The initial study of autoimmune disease prediction can be traced back to the late 20th century and demonstrated that early risk factors for inducing autoimmune responses exist in the genes of the major histocompatibility complex (MHC) . The etiEpigenetics refers to heritable changes in gene expression separate from the DNA sequence that are mediated through a series of mechanisms regulated by environmental signals . The majIn this review, DNA methylation, one of the major epigenetic adjustment mechanisms, will be reviewed, with particular attention on the function of DNA methylation in the types of cells involved in autoimmune diseases, the genomic methylation patterns involved in differentiation/development events and the dysregulated immune responses in specific autoimmune diseases. Moreover, the potential for epigenetic regulators as biomarkers and therapeutics for these diseases will be discussed.Drosophila melanogaster have been reported to lack methylation due to their undetectable m5C expression level and absence of DNA methyltransferases (DNMTs) (One of the earliest discovered (∼1969) and intensely studied epigenetic regulation mechanism is DNA methylation, which functions in producing heritable phenotypic changes without affecting the DNA sequence . Thus, u (DNMTs) . In mamm (DNMTs) . A propo (DNMTs) . In addi (DNMTs) . Moreove (DNMTs) . As one  (DNMTs) , as well (DNMTs) .de novo methylation is established during X-chromosome inactivation, and almost all CpGs in the genome are modified at that time except protected CGIs  is recruited as a methyl donor to obtain a methyl group for 5mC formation via covalent bonding . DNA metted CGIs . This brted CGIs . The DNAde novo methylation  cells can maintain their original lineage selection under the condition of stable transcriptional memory to resist redifferentiation ”  to differentiate into distinct types of memory cells and their long-term maintenance . Data fr (PMDs)”  and serv (PMDs)” . In addi17 locus . DNMT3a 17 locus . Therefo+ T cells, regulatory T cells (Tregs) play roles in limitation of inflammatory reactions and immune responses. As the “master regulator” of Tregs, Foxp3 expression is crucial for the development and function of Tregs and is present in the thymus in natural Tregs (nTregs)  stimulation. Although Tregs with DNMT1 deficiency are unable to change the methylation of CNS2 in Foxp3, global changes in DNA methylation are related to the deletion of several genes crucial to Treg function and an increase in inflammatory gene expression (As a subset of CD4(nTregs) . Conserv(nTregs) . Evidencn nTregs . In addin nTregs . Notablypression .+ T cells suggests that progressive changes in DNA methylation loss exist in the memory development of CD4+ T cells, with a linear pattern in the order of Tn-T central memory (Tcm) – T effector memory (Tem) – T CD45RA+ memory (Temra), while tissue-resident bone marrow- long-lived memory (Tmem) cells branch off with a unique epigenetic profile  committed cells  is attributed to activated B cell proliferation under the promotion of cytokines originating from Th cells and Tfh cells . Rapid pmulation . Interesmulation . Furthermulation . Notablymulation .Irf4 and Prdm-1 transcription by catalyzing H3K27me3, thereby regulating the percentage of memory B cells, GC reactions and antibody responses , CD70 (TNFSF7), CD11a , CD40 ligand (TNFSF5), and perforin (PRF1), leading to overexpression, similar to that observed in CD8+ T cells. All of these genes have a positive correlation with lupus disease activity. Furthermore, when compared with patients with inactive lupus and healthy individuals, the promoter regions of the genes mentioned above seem to be significantly hypomethylated in active lupus T cells , which is induced by oxidative stress, shows increased expression in SLE T cells and contributes to the pathogenesis of SLE. Mechanistically, CpG methylation occurs in the cAMP response element (CRE) motif, which ultimately results in hypomethylated expression of the activity of the PP2Acα promoter , consisting of 58 hypermethylated and 74 hypomethylated MVPs. The strongest T1D susceptibility genes HLA-DQB1 and GA62 (encodes GAD65), the T1D-related inflammatory cytokine TNF and the TLR receptor signaling pathway-related protein TRAF6 are representative MVPs found in this analysis; additionally, some of these MVPs were found to be altered prior to overt T1D onset and maintained temporal stability over many years, which may provide a potential possibility of early clinical diagnosis of T1D  and peripheral blood mononuclear cells (PBMCs) from fulminant type 1 diabetes (FT1D)  . CollectUNC13B, which itself is related to DN  and proliferative diabetic retinopathy (PDR) . A studyed to DN . A similed to DN . Based oo groups . In addio groups . General+ B cells, synovial fibroblasts and PBMCs; and reduced 5mC expression in synovial tissues compared with healthy controls  and CTLA-4 are associated with RA susceptibility. As important Treg signature genes, single nucleotide polymorphisms (SNPs) in Treg-specific demethylated DNA regions of these two genes can affect Foxp3 reactivation and thereby impair the normal function of Treg cells , ICAM1, MECP2, and DNMT1, all of which are regulated by DNA methylation and involved in the development of GD  and methionine synthase reductase (MTRR)] and AITD risk has been demonstrated , which include Graves’ disease (GD) and Hashimoto’s disease (HD). Recently, attention has been given to the significance of DNA methylation in GD. A genome-wide methylation analysis covering 27728 annotated CGIs and 22532 promoters in peripheral blood cells uncovered an altered DNA methylation profile in GD patients, including 132 hypermethylated and 133 hypomethylated regions. Moreover, known candidate genes that were previously identified in GD or other autoimmune diseases were also found, such as nt of GD . Moreovety to HD . In addinstrated .Over the past years, a large number of studies have explored the epigenetic regulation patterns that occur during the development of autoimmunity. As one of the three major epigenetic regulation patterns, the potential epigenetic modifications caused by DNA methylation have garnered more attention in recent years. Significantly, epigenetics can provide new insights into diagnostic and therapeutic methods for autoimmune diseases. The major potential for epigenetic application in the clinic can be summarized as epigenetic markers and epigenetic therapy. A series of epigenetic biomarkers, especially those associated with DNA methylation, are associated with clinical outcomes and provide an alternate stability profile rather than conventional testing based on DNA and RNA sequencing ; Table 1+ memory differentiation, the function of individual DNMT or TET family members in directing or maintaining CD4+ T cell memory remains to be elucidated. In conclusion, DNA methylation is a promising field that links the roles of genetics, gene expression regulation, and environmental risk factors in autoimmune diseases. To beneficially give full play to the role of DNA methylation, comprehending the definite mechanisms and critical modifications of DNA methylation and discovering strategies to alter and achieve the desired magnitude and direction of immune responses, thereby providing a potential direction for better diagnosing, monitoring and treating the progression of diseases driven by epigenetics, is essential (Although many lines of evidence have demonstrated that DNA methylation plays indispensable roles in autoimmune diseases by regulating immune cell differentiation and function, the specific mechanism by which it participates in the pathogenic states of autoimmune diseases still needs to be explored. In this review, we described DNA methylation to clarify its basic function and distribution, its ability to mediate gene expression, and the key working enzymes. Moreover, we introduced the role of DNA methylation in the development and differentiation of all types of T and B cells, discussed the controversial epigenomic differentiation models of T cells during memory development, and focused on discoveries of epigenetic control mechanisms in which the DNA methylation state is changed in both basic biological processes and the pathogenesis of a series of human autoimmune diseases. DNA methylation regulates the expression of genes that determine cell fates, predominantly via DNMTs. These genes encode key transcription factors, such as Ifng in Th1 and IL4 in Th2 cells and AICDA in GC B cells. By affecting related factor expression, dysregulated DNA methylation indirectly influences the regulatory networks in which these factors are involved, leading to amplification of effects and further deregulation of cell type-specific gene expression programmers. Although the important functions of DNA methylation in gene modification, cell differentiation and disease regulation have been confirmed, some questions still need to be clarified. For example, although it has been verified that DNA methylation plays an important role in globally controlling CD4ssential .JL performed the literature search, wrote the first draft of the manuscript, and revised the manuscript. YW, GH, XL, and ZZ critically revised the manuscript and provided substantial scientific contribution. LL and ZX proposed the project and revised the manuscript. All authors approved the final version 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Over the past decades, accumulating evidence has demonstrated a pivotal role of cholecystokinin type 2 receptor (CCK2R) in pain modulation. The established role of CCK2R activation in directly facilitating nociception has led to the development of several CCK2R antagonists, which have been shown to successfully alleviate pain in several rodent models of pain. However, the outcomes of clinical trials are more modest since they have not demonstrated the expected biological effect obtained in animals. Such discordances of results between preclinical and clinical studies suggest reconsidering our knowledge about the molecular basis of the pharmacology and functioning of CCK2R. This review focuses on the cellular localization of CCK2R specifically in the sensory nervous system and discusses in further detail the molecular mechanisms and signal transduction pathways involved in controlling pain perception. We then provide a comprehensive overview of the most successful compounds targeting CCK2R and report recent advances in pharmacological strategies used to achieve CCK2R modulation. We purposely distinguish between CCK2R benefits obtained in preclinical models and outcomes in clinical trials with different pain etiologies. Lastly, we emphasize the biological and clinical relevance of CCK2R as a promising target for the development of new treatments for pain management. Chronic pain and its management remains a major health care issue and presents a therapeutic challenge, in large part because of its complex underlying pathophysiology. During the past decade, many studies have provided evidence of a pain modulating role for the G protein-coupled cholecystokinin type 2 receptor, CCK2R. Cholecystokinin (CCK) is a peptide that was discovered in the porcine gastrointestinal tract by Ivy and Oldberg in 1928 ,4 pancreCck2r, the gene coding for CCK2R, which is located on the terminal short arm of chromosomes 11 in humans (11p15.4), is 12 kbp in length and is composed of 5 exons. According to the Ensembl database, three transcripts have been identified in humans. CCK2R splice variants and mutations have been shown to be involved in cell proliferation and cancer pathogenesis -CCK-8S labelled with Bolton-Hunter reagent to localize its binding site in DRG, led to the same conclusion [Low level expression of CCK2R has been detected in the spinal cord . Interesnclusion . Moreovenclusion , in situPain is defined by The International Association for the Study of Pain (IASP) as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage”. Neuropathic pain is defined as pain caused by a lesion or a disease affecting the somatosensory nervous system. The development of neuropathic pain arises from multiple and complex pathophysiological mechanisms making its evaluation and management difficult. Hence, gaining knowledge about pain-underpinning mechanisms and their translation into symptoms might eventually allow a more appropriate therapeutic approach to treat each patient .Since 1983, the role of CCK in hyperalgesia has been studied, in particular its interaction with opioid receptors . OpioidsMany studies have shown that CCK-8, when delivered intrathecally, via intracerebral injection or systemically, reduces the effect of opioids ,33. CCK2It is well-established that CCK is highly expressed in the hippocampus . StudiesGlutamate is the most abundant excitatory neurotransmitter in the brain, and considering its pivotal role in pain sensation and transmission, modulation of the glutamate receptor activity represents a promising potential target for pain management. As an example, the use of NMDA or AMPA glutamate receptor antagonists favorably reduce thermal and mechanical pain thresholds in both a model of inflammation and also in normal rats ,38. ThusA study using an in vitro filter wipe-off technique, revealed a modulatory role of CCK-8 on dopamine release in the rat NAc . SpecifiDopamine has a central role in pain modulation , and theAs outlined above, CCK2R activation mediates the release of dopamine, an excitatory neuromodulator, while CCK2R decreases the affinity of dopamine for its receptor D2. This mechanism could lead to pro-nociceptive effects via hyperexcitability.Pre-incubation of rat DRG neurons with CCK-8S prior to application of gamma-aminobutyric acid (GABA) resulted in attenuation of GABA-induced depolarization . This efGABA also has a significant role in pain modulation, which is suggested by the anatomical distribution of GABA receptors (GABAA and GABAB) and evidence of the action of GABA agonists on the nociceptive response. GABA receptors are present in the spinal cord and brain areas that are associated with mediating and perceiving pain ,45,46,47+ current by CCK-8/CCK2R activation has been recently shown in small-sized DRG neuron extracts from mice [+ channels that regulate membrane excitability. In DRG neurons, a decrease in A-type K+ current induces an increase in neuronal excitability and is, thus, associated with chronic pain [+ currents involves the PI3K/SRC/JNK pathway. This signaling cascade results in the phosphorylation and, thus, the inhibition of A-type K+ channels [Inhibition of the transient type-A Krom mice . DRG neunic pain . The undchannels . In the The search for and development of selective CCK2R agonists or antagonists has been a major challenge since the 1990s with potential applications spanning various medical areas. The sites and mechanisms of binding for endogenous and non-peptide ligands of CCK2R have been studied in detail, which has provided the knowledge for understanding the molecular basis of these interactions . A descrNumerous rodent models of pain have been developed and characterized in recent years. Each model is representative of one of three types of pain: neuropathic, inflammatory or nociceptive. Different mechanisms are involved according to the cause of pain. Neuropathic pain is caused by a lesion or disease of the somatosensory system, including peripheral fibers  and central neurons. Neuropathic pain can be central or peripheral, and results from trauma  or toxic injury . Inflammatory pain is caused by the release of chemicals that sensitize nerve terminals at the site of inflammation . Nociceptive pain results from normal noxious stimuli, without alteration of nerve fibers or endogenous chemical events .The role of CCK/CCK2R in central neuropathic pain was investigated in a rat model of spinal cord injury (SCI) . FollowiTraumatic injuries to the peripheral nerves can be attributed to a combination of mechanisms ranging from simple traction or stretch to ischemia, burns and electrical injuries. In general, traumatic peripheral nerve injuries impede the recovery of functions, return to work and quality of life.-/- mice did not display chronic hyperalgesia [Following chronic constriction injury (CCI) of the sciatic nerve, wild type (WT) mice displayed chronic neuropathic pain symptoms from the second postoperative day to the end of the tests (44 days), whereas CCK2Rralgesia . This stCck2r gene was upregulated after induction of the burn injury. Several behavioral tests were performed to assess the impact of the burn , the efficacy of opioid  and the effect of the CCK inhibitor proglumide on pain. Opioid drugs, injected intraperitoneally (i.p.) once daily during the experiment, partially reversed mechanical allodynia but had no effect on thermal allodynia and gait abnormalities induced by burn injury. Proglumide , significantly alleviated mechanical allodynia alone or in combination with oxycodone at low dose . Oxycodone alone at 1 mg/kg did not produce any analgesia. Co-administration of proglumide with low-dose oxycodone appeared to improve paw withdrawal force but was not statistically significant; however, it did alleviate thermal allodynia. CCK2R antagonists may be useful clinically as adjuvants to decrease opioid requirements and improve analgesic management.In a model of hind paw burn injury in mice, the involvement of CCK2R in pain  was alsoIn this study, the only CCK2R antagonist tested was proglumide, which is not a specific antagonist of CCK2R. It may have a direct interaction with δ opioid receptors, which can mask a potential effect of CCK2R blockade.Diabetic neuropathy most often affects nerves in the legs and feet. Depending on the affected nerves, painful diabetic neuropathy symptoms can range from burning, sharp or aching pain and numbness in the legs and feet, to problems of the digestive system, urinary tract, blood vessels and heart.The role of spinal CCK2R in thermal allodynia and hyperalgesia was investigated in a mouse model of streptozotocin-induced diabetic neuropathy . DiabetiThe placebo effect describes the beneficial psycho-physiological result observed after the administration of a substance or the performance of a therapeutic act, regardless of the expected intrinsic efficacy of the treatment. The placebo effect seems to be mediated by activation of μ-opioid receptors .The interaction already highlighted between CCK2R and opioid receptors demonstrates the role of CCK2R in the placebo response . The usePostoperative pain is defined as acute pain experienced by the patient after a surgical procedure. Good management of postoperative pain is important in reducing complications and facilitating rehabilitation. Postoperative pain relief is mainly based on opioids, and so is associated with side effects such as constipation, respiratory distress and the development of tolerance to administered opioids.In 1988, a clinical trial assessed the interactions between morphine and proglumide in 80 patients . All patThe use of proglumide has also been evaluated in 60 patients with dental postoperative pain . ProglumThese two studies, evaluating the efficacy of proglumide in postoperative pain, gave contradictory results, which could be explained by the use of different pain scales. Indeed, visual analogue scales (VAS) are more sensitive than category scale measurements . MoreoveCancer pain may have multiple forms. Pain can be directly caused by the tumor itself, as it grows into or destroys adjacent tissue. As a tumor grows, it can press on nerves, resulting in neuropathic pain. The tumor can also release chemicals that can cause inflammatory pain, and cancer treatments, including surgery, radiation and chemotherapy, can also cause pain.Proglumide has been tested in cancer pain in humans . A doublThe analgesic effect of the CCK receptor antagonist L365,260 on chronic human neuropathic pain that is unresponsive to current treatment, has been studied in a randomized, double-blind, placebo-controlled crossover study . Forty aMany compounds acting on CCK2R have shown promising results in preclinical models of pain. However, most of these compounds did not produce the desired effects in clinical trials. The discordance of results between preclinical and clinical studies may be explained in part by the differences in expression and distribution of CCK2R between rodents and humans. Indeed, despite a gene sequence homology of 90–97% across species , particuData are scarce concerning the exact localization of CCK2R in the nervous system, especially in the peripheral nervous system, thus limiting our understanding of CCK2R’s mechanism of action. In rodents, some information on CCK2R expression in DRG indicates a very low expression in neurons . HoweverThe lack of positive results of CCK2R clinical studies on pain management, may be due to the poor bioavailability and efficacy of administered agents . Some ofThe value of CCK2R antagonist-based therapies lies in the complex and multiple signaling activated by the CCK2R. As mentioned above, CCK2R can interact with several other GPCRs and, thus, modulate the interaction with their ligands, shifting the discharge of neurotransmitters or ion transport. These varied interactions greatly complicate understanding of the mechanism of action of CCK2R that seems to differ depending on which part of the nervous system is involved.Despite gaps in our understanding of the role of CCK2R in pain modulation, an increasing number of transcriptomic analyses underway in pain models, are highlighting the upregulation of CCK2R in several models including abdominal pain in patients with irritable bowel syndrome , a modelTo conclude, there is accumulating evidence that CCK2R plays a crucial role in the modulation of pain and that its blockade is a valid therapeutic strategy in the management of pain. Further preclinical investigations with more specific antagonists, appropriate dosing and models are needed to understand the mechanism(s) of action of CCK2R. Several promising new CCK2R antagonists have already been administered in human clinical trials with good safety profiles. Thus, these compounds represent new opportunities in analgesia pharmacology.