Major factors leading to the development of atherosclerosis are a high cholesterol (Chol) level in the blood and oxidative stress. Both promote the formation of Chol microcrystals in blood vessel walls. Deposition of Chol microcrystals in arterial intima causes inflammation, which initiates and accompanies the atherosclerotic process in all its phases. One of the possible sources of Chol in the blood vessel walls is oxidized low-density lipoproteins—this atherosclerotic plaque formation pathway has already been described in the literature. Here, we hypothesize that initiation of the atherosclerotic process may involve Chol domains in the plasma membranes of arterial cells. Increased Chol content and the presence of polyunsaturated phospholipids in these membranes together with oxidative stress (phospholipid peroxidation) may lead to the formation of pure Chol bilayer domains that, with further peroxidation and increased Chol content, may collapse in the form of Chol seed crystals. Independent of their origin, Chol microcrystals activate inflammasomes, thereby stimulate immune responses, and initiate inflammation that may lead to the development of atherosclerosis. This new, hypothetical pathway has not yet been investigated in depth; however, data from the literature and our own results support its feasibility. Biological and clinical research indicates a fundamental and initiating role of inflammation in atherosclerosis , 2. InflDuewell et al.  and RajaBoth in vivo and in vitro studies revealed that Chol crystals are phagocytosed by macrophages , 9–11. THere, we would like to discuss the plausible sources of free Chol necessary for the formation of Chol microcrystals. The presently accepted opinion is that free Chol is obtained from oxidized low-density lipoproteins (oxLDL) or other modified lipoprotein particles taken up by monocyte-derived macrophages in the arterial intima, which transforms them into macrophage foam cells , 15. An We hypothesize that the process of a Chol crystal nucleus formation is initiated in membranes containing a large amount of Chol. When the amount of Chol in a membrane exceeds the saturation limit, Chol bilayer domains (CBD) form in the membrane . A furthwhere CHC is Chol crystal.CBDs are pure Chol domains of a liquid-ordered phospholipid-Chol bilayer saturated with Chol (1:1 molar ratio). They can form in the phospholipid-Chol bilayer oversaturated with Chol. The phase of such a system is classified as a structured liquid-ordered  or dispeThe issue concerning the relationship between the Chol bilayer content and the formation of CBDs (Chol saturation limit) and Chol crystals (Chol solubility threshold) was discussed in detail in ref. . BecauseThe values of the Chol saturation limit and solubility threshold for a membrane composed of PE, PC, and SM that best represent the lipid composition of the arterial cell membrane can be estimated as the weighted sums of individual values for each phospholipid with the weight equal to the mol% of the lipid in the mixture. Such an estimate enabled us to make the phase diagram shown in Fig. cis-unsaturated acyl chain. Keeping in mind that in naturally occurring unsaturated (singly or poly) phospholipids, predominantly one acyl chain is saturated and the other unsaturated [cis-unsaturated PC-Chol bilayers is due to the reduced mixing of Chol with cis-unsaturated acyl chains in the bilayer; in effect, the Chol molecules separate from the phospholipids and form cholesterol-rich domains. The mechanisms behind this effect were analyzed in ref. [The phase diagram Fig.  was consaturated . Phosphoaturated , 36 as waturated . The low in ref. . It has  in ref. , 22 and  in ref.  are init in ref. , 39. Thu in ref. . All thiThe effects of the Chol content, lipid composition, degree of chain unsaturation, and lipid oxidation state on the bilayer phases are summarized in Fig. Molecular level processes related to atherosclerosis initiation and progression are very complex. Even though they have been studied intensively, they are not fully elucidated. The current view is that the key factor responsible for initiating atherosclerosis is LDL that undergoes oxidation by extracellular matrix proteins in the artery wall. However, clinical trials with antioxidant vitamins or folic acid in patients with preexisting atherosclerosis have proved disappointing , 41, 42 Here, we propose an alternative source of the artery wall Chol crystals potentially involved in the atherosclerosis development. We hypothesize that these crystals appear in the wall as a consequence of high-Chol content in artery wall cell membranes accompanied by peroxidation of the membrane phospholipids. As indicated in the phase diagram in Fig. Thus, according to our hypothesis, the molecular events responsible for the onset of atherosclerosis occur in the lipid matrix of the arterial cell membrane where CBDs, the precursors of Chol crystals, are formed. This hypothesis is supported by our recent experimental results , which dOur findings draw attention to the role that membrane lipids may play in controlling the direction of the CBD and Chol crystals formation process and, thus, controlling initiation of atherosclerosis development. Potential endogenic controlling factors include the lipid composition of the membranes of artery wall cells, phospholipid chain unsaturation, and the extent of phospholipid peroxidation. At present, these endogenic factors cannot be utilized for therapeutic purposes, so we can only suggest that all plausible pathways should be considered and discussed in detail to enable better understanding of the complex process of atherosclerosis initiation and progression.The connection between Chol membrane microdomains and extra-membrane Chol crystals in atherosclerosis development was first identified by Mason and Jacob . They suFor most organs and tissues, formation of CBDs and Chol crystals is considered a sign of pathology , 49. HowThe above-mentioned age-related changes occur in normal lenses , 54 and The data obtained for the eye lens and the lens fiber cells do not contradict the inflammatory nature of atherosclerosis induced by Chol microcrystals in the artery wall but they do not support the LDL oxidation pathway for formation of Chol microcrystals initiating inflammation. Finally, we want to state that the proposed pathway for formation of Chol microcrystals should be universal to any tissue where high-Chol content in membranes and oxidative stress conditions are present; and depending on the tissue, the crystals may or may not induce inflammation and disease.To conclude, we reiterate that the proposed hypothesis has already been tested in its basic sense. The published experimental results show that (1) formation of CBDs in liposomal membranes precedes formation of Chol crystals e.g., , 30, 31), 3130, 3


The proper selection of an in vitro model with a “closer to in vivo” microenvironment and structure, for instance, allows for the identification of the biomarkers involved in the early stages of glaucoma and contributes to the development of new therapeutic approaches. This review summarizes the most recent findings in the glaucoma field through the use of human two- and three-dimensional cultures. In particular, it focuses on the role of the scaffold and the use of bioreactors in preserving the physiological relevance of in vivo conditions of the human trabecular meshwork cells in three-dimensional cultures. Moreover, data from these studies also highlight the pivotal role of oxidative stress in promoting the production of trabecular meshwork-derived pro-apoptotic signals, which are one of the first marks of trabecular meshwork damage. The resulting loss of barrier function, increase of intraocular pressure, as well the promotion of neuroinflammation and neurodegeneration are listed as the main features of glaucoma. Therefore, a better understanding of the first molecular events, which trigger the glaucoma cascade, allows the identification of new targets for an early neuroprotective therapeutic approach.Glaucoma is a multifactorial syndrome in which the development of pro-apoptotic signals are the causes for retinal ganglion cell (RGC) loss. Most of the research progress in the glaucoma field have been based on experimentally inducible glaucoma animal models, which provided results about RGC loss after either the crash of the optic nerve or IOP elevation. In addition, there are genetically modified mouse models (DBA/2J), which make the study of hereditary forms of glaucoma possible. However, these approaches have not been able to identify all the molecular mechanisms characterizing glaucoma, possibly due to the disadvantages and limits related to the use of animals. In fact, the results obtained with small animals , which are the most commonly used, are often not aligned with human conditions due to their low degree of similarity with the human eye anatomy. Although the results obtained from non-human primates are in line with human conditions, they are little used for the study of glaucoma and its outcomes at cellular level due to their costs and their poor ease of handling. In this regard, according to at least two of the 3Rs principles, there is a need for reliable human-based  Primary open angle glaucoma (POAG) is a chronic disease that leads to retinal ganglion cell (RGC) loss and, consequently, the characteristic cupping of the papilla at the optic nerve level. In spite of the fact that it has been known since the time of Hippocrates, many aspects of this disease still remain obscure.The only therapy recognized to be useful for glaucoma treatment is the lowering of intraocular pressure (IOP) although the exact relationships between IOP elevation and the optic nerve head (ONH) degeneration, which leads to visual field alteration, have not been understood yet. Indeed, IOP reduction alone is not always enough for slowing-down blindness progression , 2.Many molecular mechanisms such as the ones involved in glaucoma etiology have been recognized, and in this regard, a wide range of substances with action either on a specific target or on multiple targets have been discovered .Neuroprotection, neurodegeneration, and neuroenhancement have gained great importance over time because such approaches not only prevent RGCs from death but also repair or regenerate the cell damage, changing the course of the disease .The primary molecular damage that occurs, which may be either of a degenerative/ischemic or a mechanical/metabolic nature, is able to induce changes in the extracellular matrix (ECM) of trabecular meshwork (TM) and in the TM itself that lead to retinal ganglion cell degeneration –8. The nIn glaucoma, the RGC degeneration induces trans-synaptic alterations capable of involving the entire visual chain up to the calcarine fissure . In thesTyrp1b and GpnmbR150X) it presents cause a pigment dispersion syndrome similar to that found in humans and allows for the study of the effects of elevated IOP on the retina and optic nerve head  to small animals  are used in glaucoma research. Each of these animal models can help explain some molecular aspects of such a complex disease. Generally, however, the use of large animals offers better access to eye structures compared to the use of small ones due to larger eye size. In addition, non-human primates also show a resemblance to human ocular anatomy. However, for ethical and economic reasons, small animals such as rodents have gained ground in the research community. Among rodent models, the DBA/2J mouse is widely used because the mutations  lead to glaucoma typical morpho-functional alterations such as both anterograde and retrograde RGC degeneration and trans-synaptic anterograde degeneration .Given the fact that in high-tension glaucoma the TM plays a fundamental role in its pathogenesis, several previous studies , 36–38 hTM is essential for the passage of the aqueous humor through the conventional outflow pathway. Indeed, after damage, TM cells change their gene expression  encodingex vivo and in vitro models have been used for glaucoma study. In particular, these studies have the aim to fill the gap left by animal models. The main studies are focused on the role of oxidative stress in promoting cell/tissue defects found in glaucomatous patients   , 41. Morpossible .The purpose of this review is to summarize our team's research progress using a 3D advanced human model of TM, arguing that the 2D model has limitations and hoping that our work may improve the performance in glaucoma studies.in vitro cell culture models are widely used in research to improve the knowledge about the mechanisms of disease onset and propagation and the development of preclinical drugs. However, the inconsistencies of animal researches and the oversimplification of conventional two-dimensional in vitro models have frequently delayed therapeutic advances, like in the case of glaucoma. Indeed, not all promising discoveries and treatments obtained from such models have given favorable outcomes with human evidence. Among the reasons behind animal model limitations, the poor standardization of experimental procedures and the variation of environmental conditions as well as interspecies variation  between animals are listed as the most relevant  system. Anyway, a more sophisticated question .in vivo tissue structure  provides for an up-stream analysis both of structural architecture of tissue-derived cells and of the matrices/scaffold composition in which to embed the various cell types to obtain the proper 3D microenvironment , 47, 51.In 3D cultures, matrices/scaffolds from several materials, within their structure, support cell growth, organization, and differentiation. Indeed, either biomaterials  , 53 or tin vivo environments. As is known, the interaction between cells and ECM provide all those biophysical and biochemical functions including the transport of soluble signaling molecules, nutrients, and waste metabolites as well as mechanical integrity; to a certain extent, therefore, these matrices have to reflect the features of specific tissue ECM to each application  features to better mimic lication –59. Mechlication .TM, fibrin gel, and alginate, compared to synthetic ones  are more used both because of their better biocompatibility and mild gelification -induced mechanical strain and drugs, i.e., latrunculin-B or steroids.Primary human trabecular meshwork (HTM) cells, for instance, were isolated from both juxtacanalicular and corneoscleral region, after which multiple HTM cell layers were included into a highly porous membrane, SU-8 scaffold, with predefined and well-controlled microstructure dimensions by standard photolithography techniques , 64. TheIn a study on primary porcine TM cells, high viability and proliferation after seeding into a highly porous matrix of natural biopolymer construct, the collagen-chondroitin sulfate scaffold, was reported .in vitro models of 3D HTM cells  stimulations, the upregulation of proinflammatory cytokines and MMPs after BAK treatment, and their migration through basement membranes. Therefore, the use of degradable scaffolds, compared to permanent ones, also offers an opportunity for further molecular investigations.Moreover, TM cells  and 3D mTM cells  were alsTM-based study revealed that the embedment of TM stem cell populations did not allow for cell expansion but provided the normal phenotype restoration able to express not only ESC and NC markers but also the TM ones  technology has very recently been proposed, which consisted of a high-controlled EHP system and microculture system of purified primary rat RGCs  were evaluated   were exposed to 500 μM Ht (~1 h) , 76 becaIn the previous section, we have reported the most recent results of three-dimensional culture models of trabecular meshwork (TM) and the LOC technology. In both cases, it has been demonstrated that the cells cultured in a more similar way to their native-derived tissues provide physiological responses to different stimuli.® Matrigel® Matrix  [dx.doi.org/10.17504/protocols.io.574g9qw], which consists of several proteins found in extracellular matrix (ECM) such as laminin, collagen IV, heparin sulfate proteoglycan, and entactin/nidogen  , 25 cult/nidogen .in vivo conditions, are not only cell morphology maintenance but also the continuous supply of nutrients and oxygen, as well as the removal of metabolic waste products  model and a bioreactor system, in order to overcome the issues related to cell responses under static culture conditions. In fact, the milli-fluidic technique offers precise control over gradients in a continuous manner under milli-metric-size channels , 81.in vitro model , they were subjected to recovery under dynamic conditions (In order to study the effects of oxidative stress (OS) on TM, which is one of the main causes of TM damage, TM cells were treated with daily doses of 2 h of 500 μM Hnditions , 76.in vivo as a result of the increase in outflow resistance. In our model, therefore, it was possible to measure the expression of genes related to a specific cellular activity or function after OS conditions. Indeed, quantitative real-time PCR (qPCR) assays have proved to be a useful tool for assessing the expression of individual genes in order to measure the production of mRNA encoding for both profibrotic and metalloproteinase (MMPs) markers  has partly compromised the proper identification of glaucoma biomarkers. Previous in vitro studies on TM have undoubtedly helped to understand the molecular changes in TM following OS treatment; however, most of the clinical trial results based on small animal models  revealed their translational failure mainly due to the different degree of similarity between human disease and the animal model used , 99in viin vitro model, which allows multiorgan approach through the communication between different tissues, it can be used to study the involvement of different cell types in glaucoma . In this way, it will be possible to evaluate how the TM damage, due to oxidative stress and/or increased pressure, influenced neuron homeostasis. In addition, such platform will allow to check the effectiveness of therapeutic compounds in counteracting the oxidative and/or pressure damage during the glaucoma evolution.Moreover, thanks to the features of this advanced All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Endocrine disruptors (EDs) are defined as chemicals that mimic, block, or interfere with hormones in the body’s endocrine systems and have been associated with a diverse array of health issues. The concept of endocrine disruption has recently been extended to metabolic alterations that may result in diseases, such as obesity, diabetes, and fatty liver disease, and constitute an increasing health concern worldwide. However, while epidemiological and experimental data on the close association of EDs and adverse metabolic effects are mounting, predictive methods and models to evaluate the detailed mechanisms and pathways behind these observed effects are lacking, thus restricting the regulatory risk assessment of EDs. The EDCMET  project brings together systems toxicologists; experimental biologists with a thorough understanding of the molecular mechanisms of metabolic disease and comprehensive in vitro and in vivo methodological skills; and, ultimately, epidemiologists linking environmental exposure to adverse metabolic outcomes. During its 5-year journey, EDCMET aims to identify novel ED mechanisms of action, to generate (pre)validated test methods to assess the metabolic effects of Eds, and to predict emergent adverse biological phenotypes by following the adverse outcome pathway (AOP) paradigm. The prevalence of metabolic diseases, such as type II diabetes T2D) and obesity, is rapidly increasing. According to the World Health Organization (WHO), over 460 million people suffer from T2D and 650 million adults are obese, making these diseases major health problems and a significant economic burden worldwide ,2. EspecD and obeOne of the major concerns is the human exposure to environmental contaminants, which have been shown to result in adverse health effects ,10,11,12Metabolic diseases are often associated with mitochondrial dysfunction . TherefoThe effects of hormones are mediated mainly through interactions with their cognate receptors, such as ligand-inducible, transcription-modulating nuclear receptors (NRs) ,33,34. IAdditionally, EDs may exert epigenetic modifications, such as histone modifications, DNA methylation, and the expression of noncoding RNAs ,39,40. EDespite the emerging evidence linking MDCs to adverse metabolic outcomes, as described above, the current testing tools, including regulatory in vitro and in vivo tests, are not designed to appropriately identify effects related to certain less-studied, endocrine-mediated pathways or health outcomes, in which MDCs may be implicated . New andhttps://eurion-cluster.eu). The EURION projects are focusing on different aspects of new testing and screening methods to identify Eds, and the cluster will allow the projects to optimize synergies and avoid overlaps as well as share expertise to maximize the impact of the cluster. As one of these projects, the overarching objective of the EDCMET project (www.uef.fi/edcmet) is to develop (pre)validated in silico, in vitro, and in vivo methods assessing the metabolic effects of EDs. EDCMET connects experts in various research fields, including systems toxicologists; experimental biologists with a thorough understanding of the molecular mechanisms of metabolic disease and comprehensive in vitro and in vivo methodological skills; and, ultimately, epidemiologists linking environmental exposure to adverse metabolic outcomes. To address the unmet need of validated methods assessing metabolic effects of EDs and to fill in the gaps in knowledge on less-studied ED-induced adverse outcomes, the European Commission has funded eight projects within the Horizon 2020 (H2020) framework. Together, these projects form the European Cluster to Improve Identification of Endocrine Disruptors, validated cell-based and cell-free in vitro functional profiling assays that can specifically identify MDCs and affected metabolic pathways in liver and adipose tissues. EDCMET will also provide an array of in vivo rodent tests suitable for the assessment of the metabolic effects of MDCs through the measurement of physiological functions such as glucose tolerance, dyslipidemia, liver steatosis, and obesity. Further, the project includes efforts in the identification of new tissue and plasma biomarkers, allowing for the prediction of adverse metabolic effects in vivo. Data from human cohort studies will be used to link experimental findings in animals and/or cell cultures to adverse effects in humans. In addition, the project will follow the adverse outcome pathway (AOP) paradigm to identify molecular initiating events (MIEs) and predict the emergent adverse biological phenotypes, as a thorough understanding of the mechanisms leading to adverse metabolic effects in response to EDs exposure is lacking. Thus, EDCMET aims to provide new biological prediction models that link MIEs to metabolic dysfunction (adverse outcomes). A more detailed description of individual project goals and technologies will be presented in the following sections. EDCMET will put a strong focus on NRs and the metabolic consequences resulting from their activation or inactivation by foreign compounds. However, novel and currently unidentified mechanisms will also be explored using in vitro and in vivo models utilizing unbiased omics, methods such as genome-wide approaches using next-generation sequencing techniques followed by computational methods to link MIEs to adverse outcomes. This will ultimately provide insights into the mechanisms of action of xenobiotics, which is crucial to the design of appropriate testing methods for metabolic effects exerted via the respective modes of action. EDCMET will therefore not only contribute to test development, but also to the improvement of mechanistic knowledge and AOPs .As described, many EDs have been established as MDCs and, as such, have adverse effects on metabolic health ,19,20,21Computational prediction methods aimed at predicting interactions between small molecules and macromolecular targets are widely used in drug development and predictions of toxicity . MoleculA specific challenge with identification and risk assessment of EDs is related to their varying chemical structure, interactions with numerous cellular targets, and varying effects on different physiological processes ,50. RiskThe field of toxicogenomics aims to use molecular profiling in order to establish the quantitative relationships between chemical exposure and effects on biological pathways . In partThus, we aim to collect and analyze publicly available gene expression data describing the effects of compounds in rat livers (in vivo and in vitro), human hepatocytes, and a human liver cancer cell line. Specifically, DrugMatrix , Open TGIn vitro and omics methods will be utilized to develop cell-free and cell-based profiling assays to identify biochemical and cellular responses to EDs. Further, our aim is to identify ED-affected metabolic pathways and potentially find novel AOPs leading to metabolic disorders that can be applied in future MDC screenings . To fillFirst, reference and potential MDCs will be profiled for direct interactions with NRs by applying the Microarray Assay for Real-time Coregulator-Nuclear receptor Interaction (MARCoNI) platform . The MARHepatic and adipose cells will then be exposed to high-ranking MDCs and reference chemicals and subjected to cell-based functional metabolic assays. These include functional assays such as oxygen consumption, extracellular acidification, and lipid and lipoprotein analyses, to visualize the effects at the cellular level. Similarly, RNA and protein expression will be investigated in the cell models exposed to vehicle, reference compounds, and the highest-ranking MDCs. Novel targets that do not act via NR modulation will also be identified based on expression data and gene ontologies. Finally, data from the different assays will then be compiled, and the respective assay performances will be evaluated. A ranking list for the assays will be produced and the best-performing assays will be validated with a subset of MDCs. The list of potential MDCs will be continuously pruned by the feedback from the rodent in vivo studies see  and epidEndocrine disruption typically affects processes involving multiple organs and tissues at the systemic level. Therefore, the metabolic disruption can ultimately be detected only in the whole organism or other complex systems. Although the ultimate goal for toxicity testing is the application of non-animal testing methods, it is unlikely that animal testing could be fully replaced in the near future. Studies on experimental animals, especially in rodents, are among the most common ways to identify harmful metabolic effects of chemicals, and the scientific literature frequently reports such observations . HowSusceptibility to metabolic disturbances may be modified by many individual factors. For example, an existing metabolic disease may increase the risk for ED-induced metabolic dysfunction. Therefore, we aim to investigate if obese subjects could be especially vulnerable to the metabolic effects of EDs. We aim to provide novel understanding of the mechanisms mediating the harmful metabolic effects of chemicals. This work involves use of transgenic mouse models. In the context of the AOP concept, the mode of action data should help to identify the MIEs beginning the sequence of events resulting in toxicity, the key events (KE) involved, and the final adverse outcome. In conjunction with that, we will investigate the possibility of identifying new potential plasma and/or tissue biomarkers for metabolic disruption utilizing unbiased omics techniques.In silico, in vitro, or animal models have only a minor relevance unless they are coupled with exposure data that links the levels of chemicals with metabolic endpoints in humans. The objective of the population-based assessment is to assess the levels of exposure to EDs in select cohorts in Finland, Norway, and Spain with different exposure patterns, and to associate pollutant exposure with metabolic anthropometric data as well as genetic susceptibility. A cost-effective nuclear magnetic resonance (NMR) metabolomics to interrogate metabolic parameters within existing cohorts will be employed to potentially identify novel biomarkers for metabolic effects of EDs. EDCMET has access to several relevant cohorts  that wilIn the first phase of the project, the pre-validated methods will be used to assess the concentrations of several MDCs in selected cohorts from the collected samples or from ongoing sampling at birth of newborns and mothers, in children, or adults. Low birth weight, short anogenital distance, and incidence of cryptorchidism and hypospadias have been associated with the effects of EDs in newborns, and subsequently with excess weight, high body mass index (BMI), and diabetes in adults. As described above, EDs may lead to a variety of health outcomes, such as obesity, diabetes mellitus, metabolic syndrome, alterations of female and male reproductive function (including infertility), behavioral and developmental disorders, and hormone-sensitive cancers. Identification of associations between human exposure and endocrine effects is not easy given the diversity of the environmental exposure. Therefore, the search for novel biomarkers for future use to estimate the exposure of MDCs in human populations is crucial. The level of exposure will be associated with the metabolism-related parameters collected from cohorts. Genotyping data will be used to assess the genetic susceptibility to adverse metabolic effects of EDs that can be used to identify genetically predisposed individuals. Metabolism-related parameters have been measured in a large proportion of NFBC1966 and NFBC1986 cohorts using the serum NMR platform ,71. The Knowledge gained from previous steps is crucial for the development of a study protocol suitable for detecting and following the health outcomes after exposure to ED-related compounds during pregnancy or later in life. Additionally, new MDCs identified from in silico screening for ED–protein interaction see  will be The current testing methods do not appropriately identify effects related to less-studied endocrine-mediated pathways and health outcomes but are more focused on tests and endpoints for chemicals interfering with hormones involved in reproductive functions. The developed new methods are expected to identify novel disrupted pathways in human liver and adipose tissues and to increase human relevancy in testing and risk assessment. The developed in vitro testing tools and the AOP approach are an attempt to shift from the use of laboratory animals towards the use of more human-like in vitro and computational techniques. Reducing the use of laboratory animals has a significant effect on the time and cost required for assessing toxic potential of chemicals, which makes the risk assessment economically more advantageous. Metabolic diseases are very prevalent and represent a high cost for healthcare systems in the EU countries. Our project will enhance the understanding of metabolic effects of EDs and may provide novel biomarkers for metabolic ED effects in humans. Providing better tools for the assessment of MDC effects will ultimately increase chemical safety, thereby positively impacting environmental health.The EDCMET project is expected to produce several in silico and in vitro  methods for the detection of suspected MDCs. These assays will then be forwarded for thorough validation and automation, e.g., by the Joint Research Centre of the European Union, and potentially for acceptance by the international community via the Organisation for Economic Co-operation and Development (OECD). We also aspire to complement the existing metabolic assays by omics-based techniques. In addition to yielding genome-wide data, the omics analysis of cellular and tissue samples will also generate pipeline procedures that can be used directly or after slight modification by future toxicological studies.EDCMET will gain strength from collaboration with other EURION projects that communicate best practices, in, e.g., data management and data sharing, and discuss common methodological approaches or problems in cross-cutting working groups within EURION and from EURION joint meetings where representatives of the regulatory agencies also participate. The interactions and collaborations with OBERON and GOLIATH projects that tackle the same metabolic disorders but use complementary methodologies will be especially fruitful. The use of commonly agreed EDs across many if not all assay platforms within the EURION projects should lend further validity to the findings.


The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also identified as Corona virus disease 19 (COVID-19), has recently produced a dramatic and widespread sanitary emergency. However, despite the necessity to assist a substantial number of affected patients, it is also essential to, at the same time, guarantee the usual clinical care, particularly to cancer patients, including fertility preservation (FP) strategies before the beginning of the anti-cancer treatments. The FP techniques for adult female patients include oocyte and embryo cryopreservation, which require both adequate ovarian reserve (OR) and controlled ovarian stimulation (COS) to promote multiple follicular growth. However, ovarian tissue cryopreservation is an additional FP practice suitable when an anti-cancer treatment is urgently required, whereas, for male patients, sperm cryopreservation is a simple and well-adopted procedure. Here, we focus on the current conditions in terms of agreements and rules of FP procedures during this COVID-19 pandemic to achieve and provide useful recommendations for the adoption of these techniques in patients with cancer. The diffusion of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), or Coronavirus Disease 19 (COVID-19), has rapidly become a health emergency of global proportions. The virus, primarily discovered in China to be responsible of a severe interstitial pneumonia .+ patients have a median age slightly older than 60, and male prevalence is as high as approximately 48.6%, although 25% of subjects under 45 yrs, namely in the reproductive age, are also affected, with a prevalence of a little <15% for females subjects  that Italian COVID-19+ cancer patients describe a mortality higher than 20% in patients with onco-hematological disorders, which is primarily ascribed to treatment-induced concurrent immunosuppression as a major cause influencing the cancer prognosis , in partnership with the boards of both Academic Oncologists (COMU) and the Oncology Units' Directors (CIPOMO), has recently advised specific management recommendations for oncologic patients ; they suThe innovative integrated anti-cancer treatments are undoubtedly efficient and produce improved responses with increased survival rates, thus allowing a large number of patients a chance to have a family following the cancer healing. Therefore, for these patients, the Public Health Service should warranty the availability of appropriate and urgent FP procedures in concomitance with the anti-cancer programmed treatments, even during the current COVID-19 health emergency.The International guidelines –14 for fBoth the American Society of Clinical Oncology (ASCO) and the European Society for Medical Oncology (ESMO) recommend the cryopreservation of both mature oocytes and embryos in women with an adequate ovarian reserve (OR) and after controlled ovarian stimulation (COS) to induce the multiple follicular growth (MFG). However, to avoid the hormonal stimuli, it is also alternatively suggested to cryopreserve immature oocytes after their ovarian retrieval since the oocytes are not greatly damaged by the freezing injury and are metaphase spindle free . When thConcerning the male patients, the semen cryopreservation is a largely adopted FP procedure that, if properly programmed, does not imply any delay in starting anti-cancer treatments. However, at present, several groups are interested in the optimization of testicular tissue reimplantation previously cryopreserved, although this procedure has not yet been tested in humans .The COVID-19 pandemic has brought distinctive challenges to the global healthcare community for the dramatic worldwide escalation of morbidity and mortality.+ patients based on the limited data reported in literature. In fact, despite the feared risk of infection as well as increasing morbidity and possible adverse perinatal outcomes in infected pregnant women   and the nt women , 23, vernt women . Therefont women .+ infection during the COS should, however, suggest the cancellation of the FP program, whereas when the oocyte pick-up has been already completed, the egg cryostorage is pursuable for an embryo transfer—at least until the condition is proved to be disease free. Therefore, the ART Units probably need to implement proper rules in their organization to warrant programmed treatments to COVID-19+ subjects in addition to conventional precautions and prevention measures, and, for the lack of defined guidelines, access to ART procedures has been restrained, particularly in geographic areas of high virus diffusion and morbidity as well as in several regions of northern and central Italy. Therefore, besides the utility of autonomous solutions to assist COVID-19+ people by ART centers, it is essential that both health authorities and scientific societies share and adopt defined and standardized criteria and rules.Occurrence of COVID-19During COVID-19 pandemic, the European Society of Human Reproduction and Embryology (ESHRE) recommends that all healthy and fertile women planning a natural pregnancy, even in the absence of COVID-19 infection, should avoid their maternity project at this time, while for infertile patients undergoing ART treatment it would be preferable to postpone the procedures based on oocyte or embryo cryopreservation. Furthermore, the American Society for Reproductive Medicine (ASRM) suggests, for healthy subjects, the interruption of any ART treatment, including the gamete cryopreservation, as well as the suspension of embryo reimplantation and elective surgery, while maintaining the cryopreservation procedures for cancer patients urgently undergoing COS .In prepuberal and adult patients affected by hormone-sensitive cancers and urgently requiring gonadotoxic protocols, the ovarian cortex cryopreservation for future reimplantation appears to be a suitable procedure to prevent the delay of cancer treatment, though COVID-19 testing appears to be necessary for patients before addressing the pelvic surgery to obtain the ovarian tissue. Similarly, the ovarian transposition, a FP procedure suggested before starting pelvic radiation therapy, should require COVID-19 diagnostics before its application in females with cancer, whereas the temporary ovarian suppression of GnRH agonists during anti-cancer treatment is a protective procedure for the ovarian function and is also pursuable during this pandemic once COVID-19 testing has been assessed in patients .+ cancer males do not contain the virus and that their cryopreservation could be regularly adopted for FP programs.However, based on the short time since the COVID-19 emergence, a major question concerns the few data disseminated from the relevant scientific societies on the proposal of a shared consensus on either the prosecution or interruption of ART procedures in cancer patients. There should be, indeed, no reason to consider them at a higher of infection once well-defined precautions have been adopted. On the other hand, the postulated risk of disease transmission in gametes by infected people has not been definitely assessed, and, at least in males, the presence of the virus in seminal fluid has not been confirmed , thus suBy contrast, there are no data evaluating the risk of a possible transmission of COVID-19 to oocytes in infected women after their COS and oocyte recruitment following the MFG. Therefore, the lack of information related to this specific topic cannot support or deny the suitability and safety of ART procedures in infected cancer females.+-related issues , and two more babies died for other reasons; none of the newborns died from COVID-19d issues . News fr+ cancer patients to transfer the infection to their own gametes, and more studies are necessary to assess these questions in planning the FP activities to prevent the viral transmittance to fetuses.Nonetheless, the available data are inadequate to support the wished inability of both males and females COVID-19It has been reported in literature that the initial symptoms of the COVID-19 infection, such as cough, fever, shortness of breath, muscle pain, chest pain, headache, and diarrhea, are similar to those occurring in cancer patients after treatment protocols using chemo, targeted, and immune therapy , and patIn fact, it has been reported by Liang et al. that the risk of COVID-19 infection is definitely higher in cancer patients as an effect of their clinical condition of increased morbidity, which rapidly result in worsening and the occurrence of severe complications requiring intensive therapy care, especially as compared to other non-cancer patients .Therefore, it is absolutely necessary during the FP counseling for cancer patients to triage for COVID-19 in accordance with local guidelines, which also includes a nasopharyngeal buffer for the screening of the infection for its potentially impact on the prognosis. However, at present, despite the absence of studies investigating cancer patient candidates to FP procedures of a fertile age, this standardized screening procedure, which is largely used today when facing the infection, should be performed.The FP is an essential opportunity for cancer patients requiring gonadotoxic treatment and the desire to produce a family following cancer recovery.+ cancer patients, the virus transmittance to either sperms or oocytes is apparently an averted biologic event, and the cryopreservation of gametes can be maintained in these patients following the necessary precautions against the infection. In these situations, it is critical to have dedicated triage and to respect all the imposed limitations to avoid the increase of the infection. On the other hand, it is also necessary for all ART and other centers enrolled in FP programs to adopt uniformly regulated rules when managing cancer patients undergoing those procedures to avoid the risk of infection or reveal its presence in apparently asymptomatic patients. To this, it is also necessary that health authorities and scientific societies take an interest in this topic and provide regulatory standards.Therefore, despite the very few published data on the FP topic in COVID-19In conclusion, while it is possible to reassure these patients that their gametes are not infected by COVID-19 and can thus be processed for cryopreservation, a screening by serum detection of antibodies and COVID-19 proteins, or a nasopharyngeal pad, should be mandatory in cancer patients participating in FP programs.MD reviewed the literature and wrote the manuscript. CM contributed to literature review. AP contributed to writing. RD and ES contributed critically reviewed the manuscript. ES reviewed the literature and wrote the manuscript. All authors have read and agreed to the published 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.


To date, across the globe, over 45 countries, cities, and regions have instituted sugar-sweetened beverage (SSB) taxes. We see at least 2 major health-related reasons to focus on SSBs. First, excess sugar consumption is a major cause of obesity and the increasing risk of type 2 diabetes, hypertension, liver and kidney damage, heart disease, and some cancers . Second,PLOS Medicine highlight the potentials of different methods of discouraging SSB consumption among the public and encouraging reformulations by the beverage industry. In the United Kingdom, the multitiered Sugar Drinks Industry Levy based on sugar content has prompted remarkable reformulations and shifts in purchases with new low-calorie beverages emerging [Three recent papers published in emerging . In Portemerging , and theemerging . Likewisemerging . Taillieemerging . While rOverall, price changes are heterogeneous depending on the baseline levels of consumption, market shares of beverage brands given the geographic coverage of the tax, and reflect strategic behaviors by beverage companies and retailers ,21. ConsThe findings to date suggest that future SSB tax designs need to consider the baseline levels of consumption of various beverages stratified by income and the price elasticities of demand that can guide the scope of the products covered. Second, the tax structure should be aligned with the primary objectives. If the goal is reduction of sugar consumption, then one based on sugar density is more likely to achieve the goal, as the Portugal and UK results suggest. If the goal is revenue generation, then a volume-based specific tax across a broad scope of beverages may result in greater revenue given a weaker incentive to reformulate. Meanwhile, studies have shown that ad valorem taxes on SSBs are less likely to be fully pass through onto prices compared to specific taxes in the form of sales taxes (rather than excise taxes) . FinallyA critical concern from the health perspective is the reductions in sugar and caloric intakes through reduced SSB consumption. While these taxes specifically affect high consumers ,26, evalEvidence to date demonstrates that sugar reduction policies will and have resulted in the introduction of beverages with both sugar and nonnutritive sweeteners (NNSs) ,16,33–36NNS consumption is growing in high-income countries, but it is less clear what will happen in low- and middle-income countries, where consumption of diet beverages is minimal . The MexSo why the concern around NNSs? One fear is the impact on sweetness preference and habituation among children. Among adults, we see different outcomes in widely conflicting human studies looking at gut health, brain response, and heart health –43. At pA few Latin American countries are exploring front of package labeling to inform consumers if products contain NNSs. Additional information on the amounts of the various NNSs will allow monitoring of our exposure to these additives and population-based observational studies on health outcomes of the types and amounts of NNSs in diets. Currently, Chile is the only country, to our knowledge, that requires the amount of each type of NNS on nutrition labels.Of course, the food industry constantly undertakes research and development, and the scientific community’s understanding of how the various combinations of foods, ingredients, and chemicals we are exposed to affects our health over time is still growing. Any new regulations targeting current attributes of concern will meet subsequent introductions of new ingredients and products to avoid or minimize such regulations. Researchers and regulatory agencies must be vigilant and thoughtful in establishing mechanisms with which to periodically assess and improve these regulations to ensure that they evolve with the food landscape to best protect people’s health.Taxation of SSBs is an important start to using fiscal policy to correct the large human and planetary costs of the modern food supply chain and promote improved diet and eventually health . SSB tax


For social animals that communicate acoustically, hearing loss and social isolation are factors that independently influence social behavior. In human subjects, hearing loss may also contribute to objective and subjective measures of social isolation. Although the behavioral relationship between hearing loss and social isolation is evident, there is little understanding of their interdependence at the level of neural systems. Separate lines of research have shown that social isolation and hearing loss independently target the serotonergic system in the rodent brain. These two factors affect both presynaptic and postsynaptic measures of serotonergic anatomy and function, highlighting the sensitivity of serotonergic pathways to both types of insult. The effects of deficits in both acoustic and social inputs are seen not only within the auditory system, but also in other brain regions, suggesting relatively extensive effects of these deficits on serotonergic regulatory systems. Serotonin plays a much-studied role in depression and anxiety, and may also influence several aspects of auditory cognition, including auditory attention and understanding speech in challenging listening conditions. These commonalities suggest that serotonergic pathways are worthy of further exploration as potential intervening mechanisms between the related conditions of hearing loss and social isolation, and the affective and cognitive dysfunctions that follow. Social isolation is a major concern among people with hearing loss ,2,3,4. UThe loss of input from the auditory periphery triggers a cascade of plasticity within the central auditory system, multiple aspects of which have been extensively reviewed. Following peripheral loss, the central auditory system undergoes a progressive rebalancing of excitation versus inhibition and synaptic plasticity that encompasses both ascending and descending systems  that show l bodies , or evenl bodies . The denl bodies . Tract tl bodies ,81,82. Ml bodies ,85,86,87Within this anatomical framework, serotonergic function within the auditory system has been assessed by directly measuring serotonin availability during different kinds of events, and by manipulating serotonin or its receptors, and subsequently, monitoring neural or behavioral responses. The direct measurement of serotonin in awake and behaving rodents has shown that serotonin increases in response to both acoustic and non-acoustic stimuli. The presentation of white noise increases serotonergic activity in auditory regions caudal to the thalamus ,90. BothIn the auditory midbrain, serotonergic activity is also elevated during multiple types of social interaction: in males with either male or female social partners, and in females with male social partners ,63,92. IThe responsiveness of auditory neurons to fluctuations in serotonin has been assessed by bypassing endogenous patterns of serotonin release and directly manipulating serotonin or specific types of serotonin receptors within the auditory system. These kinds of manipulations have effects that are observed at the level of individual neurons, specific neural circuits, and neuron populations. The effects of these manipulations depend on the cell type being monitored and the serotonergic receptor type that has been activated or blocked. For example, in the inferior colliculus (IC), 5-HT2A receptors facilitate inhibitory transmission by presynaptically increasing the frequency of spontaneous inhibitory postsynaptic events . SerotonAt the level of the neural population in the IC, manipulating serotonin availability alters the number of neurons expressing the activity-dependent immediate early gene product cFos in female mice . The effGiven these effects of serotonin within many regions of the auditory system, it is hardly surprising that serotonin can influence auditory processing and acoustic perception in human subjects. High levels of serotonin have been associated with a shallower slope of Wave V and the N1/P2 potential of auditory-evoked responses ,97,98, aSummaryPresynaptic and postsynaptic elements of serotonergic pathways are present throughout the auditory system. Serotonin responds to broad classes of events such as stressful events or social interaction. Within the category of social interaction, serotonergic release parallels the salience of specific interactions. Once released, serotonin has cell-specific and receptor-specific effects that functionally reconfigure auditory circuits. Given the scale of serotonergic innervation of the auditory system and the ability of serotonin to induce plasticity in auditory processing, it is striking that the serotonergic system itself exhibits extensive plasticity. Plasticity can result from long-term changes in acoustic input, but also occurs in response to changes in the general social environment. This type of sensitivity to environmental influence can serve an important function in adapting serotonergic regulation to long-term changes in internal and external conditions. Plasticity in serotonin–auditory interactions is expressed at both presynaptic and postsynaptic mechanistic levels. That is, changes occur in both the serotonergic fibers and the dynamics of serotonin release, and in the expression of serotonin receptors by auditory neurons.Sound-induced hearing loss alters the densities of serotonergic fibers and the functional dynamics of serotonin release and reuptake. Following monaural exposure of mice to an 8 kHz tone at 113 dB, axons that are immunopositive for the serotonin transporter (SERT+) in the inferior colliculus (IC) are altered in density . SerotonFollowing bilateral tonal acoustic trauma of rats, densities of SERT+ axons are also lower in multiple auditory regions, including the cochlear nucleus, IC, and auditory cortex relative to a non-trauma group . In paraPostsynaptic changes in serotonergic pathways are also altered by hearing loss. Following hearing loss induced by bilateral exposure to a 10 kHz tone at 116 dB, the expression of multiple types of serotonin receptor are altered in the IC. Receptors in the 5-HT1 family, such as the 5-HT1A and 5-HT1B receptors, are strongly expressed in the IC ,84,85. ASpeculation about the functional consequences of these changes in gene expression can be supported by the distinct roles of these receptor types. The 5-HT1A receptor is often expressed somatodendritically or in the axon initial segment, and often suppresses neural firing ,59,121. Changes in the expression of 5-HT receptors are also observed after hearing loss associated with age or following cochlear ablation ,87. The Some of these effects of exposure to loud sound on the serotonergic system could occur in part through the activation of stress-responsive physiological systems rather than through a primary effect on the auditory system. Noise exposure itself is a stressful event that can increase serum glucocorticoid levels and alter the expression of glucocorticoid receptors, although these effects are dependent on the type of stressor and brain region ,124. ThrSummaryHearing loss is associated with presynaptic and postsynaptic changes in the serotonergic system within auditory regions, but some of these changes also occur in other brain regions. Stressors associated with the induction of hearing loss may independently contribute to the plasticity in local serotonergic pathways.As a factor on its own, social isolation has significant consequences for auditory function. While some work has demonstrated an effect of social interaction on auditory responses in humans , the buAt the level of single cells, the response properties of individual auditory neurons change in ways that suggest that auditory processing is more finely tuned in socially housed birds. Auditory neurons of socially housed birds show less noisy, more precise responses to sound compared to isolated birds ,44. FurtSocial isolation also alters the neural processing of sound on a larger scale. For example, when compared to socially housed birds, birds raised in isolation have more responsive sites across an avian auditory forebrain area, Field L ,44. ThisIn fact, a few studies suggest that social isolation also shapes auditory perception. For example, completely socially isolated birds of several species are unable to discriminate between songs based on frequency; socially deprived birds are also unable to discriminate between different song notes ,49,50. IWhile deprivation of social stimulation alters auditory responses on their own, social deprivation also affects the integration of non-auditory sensory input with auditory processing. In birds raised with adults, images of familiar versus unfamiliar birds differently alter auditory-evoked activity . In contResearch in songbirds suggests that social isolation itself influences auditory processing, but whether this phenomenon extends to other taxa, such as mammals, is relatively underexplored. However, there is a strong body of literature in mammals, particularly in laboratory rodents, demonstrating that social deprivation causes dramatic effects on behavior ,67,135, In addition to the research on the acoustic startle response, one study has explored whether social experience shapes auditory perception in mammals, by investigating the influence of social versus individual housing on auditory discrimination in mice . HousingSummarySimilar to hearing loss, social isolation induces plasticity in auditory processing and may influence the integration of acoustic and non-acoustic information within the auditory system. The majority of this work has been conducted using songbirds as a model system. Future studies should investigate whether social deprivation of mammals causes similar deficits in auditory processing.The serotonergic system provides a source of non-acoustic input to the auditory system and is sensitive to the social environment; it is therefore one potential pathway through which social isolation could influence auditory function. Whether social isolation reconfigures this pathway is just beginning to be explored. Two studies ,147 suggIn the Keesom et al.  study deAlthough there is no effect of social isolation in males on the density of SERT+ projections to the IC, depriving male mice of social contact does alter directly measured serotonergic activity in the IC triggered by the presence of a social partner . In thisSummarySocial isolation influences the interaction between the serotonergic and auditory systems, with effects on serotonergic fiber density and serotonergic activity in the auditory midbrain that match changes in other factors sensitive to isolation, including body weight and behavior.Although this synthesis paper has focused on the auditory system, hearing loss and social isolation both influence the serotonergic system outside of auditory regions. This has been best explored for social isolation. Social isolation influences serotonergic axons, activity, and metabolism throughout the brain, including regions involved in cognition, memory, emotion, and reward. In comparison to the sex-specific decrease in serotonergic fiber density that it causes in the auditory system, social isolation has mixed effects on serotonergic axons across several extra-auditory brain regions. Isolation decreases serotonergic fiber density in the hippocampus  but incrThe extra-auditory effects of both hearing loss and social isolation are particularly interesting in light of the important role of interaction among auditory and non-auditory regions in giving rise to perceptual dysfunction following hearing loss. For example, relative to human subjects with equivalent hearing loss, subjects with tinnitus have anatomical and functional differences in brain regions, including the ventromedial prefrontal cortex and the amygdala ,169,170.The interactive effects of hearing loss and emotional systems on perception are also illustrated by a three-way interaction between hearing loss, the subjective perception of social isolation, and the affective perception of acoustic stimuli. Subjects with hearing loss show dampened emotional ratings of affectively charged sounds, use a smaller range of values to describe such sounds, or take longer to respond to affective sounds but not neutral sounds in comparison to normal hearing listeners ,177,178.SummaryHearing loss and especially social isolation influence presynaptic and postsynaptic elements of the serotonergic system outside of the central auditory system, including in brain areas related to memory, reward, and emotion. This is particularly interesting given that alterations in the functional connectivity between auditory and non-auditory regions are associated with deficits in the perception of acoustic stimuli, and that the perception of social isolation co-occurs with an altered emotional response to sound.Although the current paper presents evidence that hearing loss and social isolation each influence the serotonergic system, whether their effects actually converge on the same targets, and if so, at what levels of organization, have not yet been experimentally explored. For example, multiple subgroups of serotonergic neurons with different responsiveness to external events may innervate the same auditory regions ,82. It iThese uncertainties allow for the possibility of significant nonlinearity in the interactions between hearing loss and social isolation at the level of auditory processing and perception. Possible types of interaction include synergy, in which the effects of hearing loss and social isolation are greater than either alone. In this model, multiple separate ‘hits’ to the serotonergic system caused by hearing loss and social isolation, could create extreme dysregulation of auditory processing. Conversely, maintained social connection could minimize negative effects of hearing loss on serotonin–auditory interaction. This could fall within the ‘social buffering’ hypothesis, in which social contact counteracts some of the negative effects of stressors . In contOn a broader scale, the possibly convergent effects of hearing loss and social isolation on the serotonergic system play out within the context of many interacting sensory and cognitive systems that may themselves be sensitive to these factors. A useful framework for envisioning different types of interactions among auditory and non-auditory systems is the increasingly supported link between hearing loss and cognitive decline in older adults. Difficulty in hearing is predictive of cognitive deficits in a range of tasks assessing memory, executive function, and speed of response ,189,190.The idea that social isolation itself may play an important role in cognitive decline following hearing loss is supported by a recent study modeling the causal interactions of hearing loss, social isolation, and subjective loneliness in cognitive outcomes . The stuModels of the links among hearing loss, isolation, and perceptual or cognitive outcomes that incorporate physiological systems can serve an important role in illustrating the potential for ameliorating some of the consequences of hearing loss and social isolation. For example, some of the hypotheses linking hearing loss with cognitive decline predict that assistive hearing devices should prevent or reduce cognitive deficits. There is increasing evidence that such sensory interventions do decrease cognitive declines, or slow the rate of decline e.g., ,209,210),210209,2Social animals such as rodents, songbirds, and humans may have the opportunity to adapt their behaviors to a wide range of social environments, from enriched to impoverished networks of social contacts. This capability may involve neuromodulatory systems, such as the serotonergic system, given that serotonergic pathways respond to sensory stimuli and external events and are also highly attuned to the social environment. These functions fit the description of a system that could respond to comorbid conditions such as hearing loss and social isolation. Disruptions in neuromodulatory function due to hearing loss and/or social isolation may also have consequences related to the role of these same neurochemical systems in regulating intrinsic neural processing in many brain regions, as well as in regulating connectivity among interacting networks. These dual roles make these systems, including the serotonergic system, a potentially valuable key for understanding the origins of links among social environment, sensory function, communicative dysfunction, and cognition.


It is well known that a gram of soil contains thousands of individual microbial taxa including bacteria, fungi, protists, oomycetes and viruses. Many of them play the main role in ecosystem functioning determining soil fertility and provide plant growth promotion and disease suppression, . HoweverThere are many interrelated points in our understanding of the role of PBM that should be taken into consideration when designing inocula of PBM and applying them in the field. Firstly, the coexistence of all multicellular eukaryotes and microorganisms forming a holobiome and hologenome was evolutionary proved. The vast majority of recent studies including in the field of plant–microbe interactions, have confirmed the role of beneficial microorganisms in host development, metabolism, stress adaptation, and health. It appears that hosts can attract microorganisms with specific plant-beneficial characteristics . SecondlBased on the above considerations, three strategies for microbial management of soil–plant systems could be selected based on prebiotics, probiotics, and postbiotics Figure 1Prebiotics are products, which improve microbial diversity and soil microbial health by promoting the growth of soil microorganisms already present within the soil–plant system. Prebiotics are natural products, normally agro-industrial wastes, including biochar, sewage sludge, compost, humus, animal manure, and chitin-bearing wastes, among others, which ameliorate (particularly in degraded soils) the soil structure, biochemical activity, and increase microbial population and diversity . CompostIn the field of soil–plant science, probiotics are accepted as beneficial microorganisms, which exert health promoting and nutrient-mobilizing properties, as defined by Postbiotics are metabolic derivatives of PBM, which exert specific, growth promoting and/or biocontrol, effects on plants thus avoiding the risks associated with applying microbial cells. Specific examples of such metabolite include phytohormones, volatiles, and quorum-sensing compounds . Which aShall we apply cell-free liquids containing specific or complex metabolites produced by the PBM during fermentation under controlled conditions? There are two options in developing such kind of biotechnological products. Using cell-free fermentation broth liquids without further downstream operations for separation/purification of specific metabolites is the most economic option and, in some cases mixtures of different microbial cultures demonstrate higher potential even after autoclaving . Well-esAnother approach includes operations such as fragmentation and further use of extracts of the microbial mass or isolation of specific metabolites from the fermentation liquid. However, the application of specific metabolites in soil should be assessed carefully, bearing in mind that in the rhizosphere there is a great variety of microbial and plant metabolites involved in a wide number of interrelated cooperative or antagonistic actions . TherefoProduction and application of PBM is now one of the most promising fields of research. The period of searching for easy to cultivate soil microorganisms, their characterization, and testing in controlled conditions was replaced by another one with studies on novel, more efficient and economic fermentation mode of production and formulations. Co-cultivation and formulation of compatible PBM and inclusion of various additives in the formulations become fundamental part of the overall production technology . AnotherMV and NV designed and drafted the work. EM and EF-P contributed to the revision of the manuscript.This work was supported by the project EXCALIBUR funded from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 817946.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.


Enterobacteriaceae family, is one of the envelope stress response pathways. The Rcs system can sense envelope damage or defects and regulate the transcriptome to counteract stress, which is particularly important for the survival and virulence of pathogenic bacteria. In this review, we summarize the roles of the Rcs system in envelope stress responses (ESRs) and virulence regulation. We discuss the environmental and intrinsic sources of envelope stress that cause activation of the Rcs system with an emphasis on the role of RcsF in detection of envelope stress and signal transduction. Finally, the different regulation mechanisms governing the Rcs system’s control of virulence in several common pathogens are introduced. This review highlights the important role of the Rcs system in the environmental adaptation of bacteria and provides a theoretical basis for the development of new strategies for control, prevention, and treatment of bacterial infections.The bacterial cell envelope is a protective barrier at the frontline of bacterial interaction with the environment, and its integrity is regulated by various stress response systems. The Rcs (regulator of capsule synthesis) system, a non-orthodox two-component regulatory system (TCS) found in many members of the  The cell envelope of Gram-negative bacteria is generally composed of an inner membrane (IM), a periplasm with a thin peptidoglycan layer, and an outer membrane (OM)  Figure . The OM E response system, Cpx response system, Bae response system, Psp response system, and regulator of capsule synthesis (Rcs) response system are known to sense and respond to cell envelope assaults  the Rcs system is the only signal transduction system known to have an OM component (RcsF) that senses almost all induction cues ; and ii. NumerouEscherichia coli  is an IM protein that seems to function by inhibiting the Rcs signaling, thus ensuring that the signal through this phosphorylation is minimal in the absence of environmental stimuli  domain in an ATP-dependent manner. The phosphoryl group is then transferred to the aspartate residue Asp875 on the phosphoryl receiver (PR) domain of RcsC. The phosphoryl group is subsequently transferred to the histidine residue His842 on the histidine-containing phosphotransmitter (HPT) domain of RcsD, and finally to the aspartate residue Asp56 on the PR domain of RcsB -mediated damage during infection  mutation and ompA deletion  can affect peptidoglycans, thereby inducing the Rcs system. For instance, osmoregulated periplasmic glucans (OPGs) defects caused by opgGH deletion in Yersinia enterocolitica , periplasmic signals that perturb peptidoglycan, and lipoprotein mislocalization caused by environmental stress or intrinsic sources of stress lead to the activation of the Rcs system, which in turn regulates its target genes to allow cells to adapt to environmental and genetic changes.RcsF is an OM lipoprotein that contains a lipidated N-terminal membrane-anchored helix followed by a 30-amino acid proline-rich linker and a well-folded 87-amino acid periplasmic domain . RcsF loS. enterica for its effect on intracellular growth and virulence , this information is transduced to the RcsF C-terminal signaling domain located in the periplasm to activate the stress response . In addition, the Rcs system also regulates genes involved in the assembly of the secretion system and proteins that have predictive effects on cell surface maintenance and modification. Therefore, it is generally believed that the Rcs system affects bacterial motility, biofilm formation, intracellular survival, and invasiveness . The RcsS. enterica serovar Typhimurium, a principal agent of gastroenteritis in humans  can reduce the virulence of S. enterica in mice. This virulence attenuation depends on the RcsB phosphorylation level and partly depends on RcsA and colanic acid production  was also shown to reduce the phagocytosis rates of Salmonella by murine macrophages, and this defect might be attributed to an increase in colanic acid production  or negatively (high RcsB-P levels) regulate the expression of the SPI-1 and SPI-2 pathogenicity island genes , which is important for persistent S. enterica serovar Typhimurium infection in mice  O157: H7 can cause hemorrhagic colitis with a low minimum infectious dose  (Enterohemorrhagic ous dose . In thism (T3SS) . The incm (T3SS) .E. coli K12  study of avian pathogenic E. coli-mediated septicemia in chickens , thereby increasing the translation level of the rpoS mRNA  Yersinia pseudotuberculosis, a zoonotic pathogen of mammals and birds that occasionally causes enterocolitis, mesenteric lymphadenitis, septicemia, and immune-mediated diseases in humans; (ii) Yersinia pestis, the causative agent of plague, including the medieval “Black Death”; and (iii) Y. enterocolitica, the Yersinia species most frequently associated with human infections. The Rcs system has been proven to regulate the virulence of the three bacteria , which increases the Yop effector protein secreted by the bacteria in the host immune cells, thereby enhancing survival of the bacteria  by regulating the CPS-related genes; further, the Rcs system in Klebsiella pneumoniae repressed the expression of fim gene cluster, which could acts as a virulence factor that facilitates the urinary tract infection  in the absence of RcsF, the Rcs system continues to function normally. For RcsF-independent signals, the mechanism by which the Rcs system senses signal molecules and achieves signal transduction remains unknown; (ii) to date, most studies have identified Rcs-regulated genes under in vitro growth conditions or by over-activating the Rcs system, and few studies have performed genome-wide gene expression under in vivo conditions. Owing to the differences in the regulation of gene expression by the Rcs system in vivo and in vitro, it is necessary to conduct transcriptomic studies of the Rcs system in vivo to identify new virulence-related target genes. In summary, the Rcs system provides a unique model for studying the complexity of environmental adaptation in bacteria. An understanding of the Rcs system will help to provide a theoretical basis for the development of control, prevention, and treatment of bacterial infections.The Rcs system is an important signal transduction pathway found in many members of the JM, GY, and JC wrote the manuscript. All authors have approved the final version of the manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: The World Health Organization has reported more than 31,186,000 confirmed cases of coronavirus disease-19 (COVID-19), including 962,343 deaths, worldwide as on September 21, 2020. The current COVID-19 pandemic is affecting clinical research activities in most parts of the world. The focus on developing a vaccine for SARS-CoV-2 and the treatment of COVID-19 is, in fact, disrupting many upcoming and/or ongoing clinical trials on other diseases around the globe. On March 18, 2020, the United States Food and Drug Administration (FDA) issued an updated guideline for the conduct of clinical trials during the current health emergency situation. The potential challenges, such as social distancing and quarantines, result in study participants’ inaccessibility and trial personnel for in-person scheduled study visits and/or follow-up. Due to the sudden onset and wide-spread impact of COVID-19, its influence on the management of clinical trials and research necessitates urgent attention. Therefore, our systematic review of the literature aims to assess the impact of the COVID-19 pandemic on the conduction of clinical trials and research. The search for the relevant articles for review included the keywords “COVID-19” AND “clinical trial” in PubMed, MEDLINE, Embase, Google scholar and Google electronic databases. Key findings include: delaying subject enrollment and operational gaps in most ongoing clinical trials, which in turn has a negative impact on trial programmes and data integrity. Globally, most sites conducting clinical trials other than COVID-19 are experiencing a delay in timelines and a complete halt of operations in lieu of this pandemic, thus affecting clinical research outcomes. The various preventive measures adopted by different countries included travel restrictions , social distancing, with the closure of schools, restrictions on mass gatherings, quarantine, adherence to personal hygiene, use of face masks and, the implementation of infection control measures in healthcare settings [The World Health Organization (WHO) declared coronavirus disease-19 (COVID-19) a global public health emergency in late January 2020. COVID-19 is a newly identified coronavirus strain that initially infected people in Wuhan, China [th of March 2020, the US (United States) Food and Drug Administration (FDA) issued new guidelines for clinical trials’ conduction during this critical situation. The significant obstacles encountered are ‘created’ by social distancing and quarantine measures which forbid interaction between the study participants and trial personnel for study visits and scheduled follow-up. The clinical trial’s integrity is questionable due to the spread of COVID-19 infection to trial participants and staff, which influences trial outcomes with increasing likelihood of trial dropout. Finally, there may be limited access to beds, clinical tests, and Personal Protective Equipment (PPE) as these might be diverted to clinical care facilities and staff to deal with COVID-19 patients.The monitoring of patients is possibly the most significant ongoing challenge that the performing trials sites are currently facing. On the 18Due to the sudden onset and widespread impact of COVID-19, its influence on managing clinical trials and research remains undetermined . TherefoThe current systematic review process was performed and reported following the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). The proCOVID-19” AND “clinical trial”, which were fully indexed in PubMed, MEDLINE and Embase. Furthermore, in order to search for additional eligible records, a similar search strategy was adequately deployed in Google Scholar and Google. The keywords used for searches were variants representing the term coronavirus such as “2019-nCoV”; “2020-nCov”; “ 2019-20 coronavirus*”; “ 2019-2020 coronavirus*”; “SARS-CoV-2”, AND “clinical trial” mentioned either in the title or in the abstract. We have included and screened all published articles highlighted the impact of COVID-19 on clinical trials and clinical research as there is a paucity of literature on this topic due to the sudden onset and evolving nature of the COVID-19 pandemic.This study’s eligibility criteria were articles primarily focusing on the thematic interface between “The included studies were (a) published original research, (b) written in English; (c) completed and published between January 1, 2020, and September 05, 2020; (d) highlighted the impact of COVID-19 on clinical trial; (e) focused on patients from the global listed countries.The decision to include or exclude a study was based on the availability of information regarding the impact of COVID-19 on the clinical trial. The relevant studies available as full texts for review were further considered in this review; whereas, abstracts without full texts were excluded.The results of the searches of the databases were initially scanned for appropriate titles of the studies. The selected titles were then screened for the abstracts as well as full texts, and those that met the eligibility requirements were considered for final selection. All the literature evaluation (including abstracts and full-text articles or reports) was independently conducted by four researchers . The extracted data mainly included the study’s origin, authors, sample size, study population, design, settings/location, period and outcome.All systematic reviews incorporate a process of critique or appraisal of the research evidence. The purpose of this appraisal is to assess the methodological quality of a study and to determine the extent to which a study has addressed the possibility of bias in its design, conduct and analysis. We used the Studies The Joanna Briggs Institute (JBI) Critical Appraisal Checklist for Analytical Cross Sectional studies . [Table 1 [3. Overall quality was high in the studies found; nevertheless, the domains of confounding and strategies to deal with confounding factors were deficient in most of the studies because of presence of some difference between the subjects.The literature searches generated a total of 554 million articles, of which 553,999,915 were either review articles or duplicate publications that were excluded from the initial screening. Therefore, only the relevant titles and abstracts underwent a detailed assessment, and later on, 81 articles were excluded from the review process that finally resulted in six relevant articles (four research reports and two articles) included in this review. We used the PRISMA diagram to depict the flow of information through the different phases of the systematic review, it maps out the number of records identified, included and excluded , globally there is around 30% decrease in enrollment of new subjects entering trials by the end of June which was previously higher in April, 2020 (~70% drop). Currently, within different geographic regions, a marked variability has been observed regarding the extent and timing of recovery of patients due to continued fluctuation in the COVID-19 cases, and adoption of regulations over the time [Currently, the worldwide impact of COVID-19 on the conduction of clinical trials is being monitored by Medidata. The initial insights and facts about the impact of COVID-19 were reported on March 23the time . By the th, 2020  in clinical trials per study-site year-on-year (YoY) in the initial two weeks of May 2020 as compared to the same period in 2019. This impact on the average number of new subject enrollment for the initial two weeks of May 2020 is comparable to the previous month (April), showing a continued impact of COVID-19 on the new enrollment and clinical trial activities as a whole [The most recent report was released on May 15Table 3 highlighted the special considerations regarding different phases of clinical trial conduction in the coronavirus pandemic. In summary, mitigation efforts such as self-isolation and inaccessibility of health care facilities during pandemic interferes with all aspects of clinical trials at various levels. So, the delivery of intervention and collection of outcome data should requires thoughtful considerations. Finally, conduction of clinical trial activities may require suitable protocol amendments to ensure the rights, safety, and wellbeing of the study participants as well as the research staff.The ongoing COVID-19 pandemic has a potentially negative impact on the management of clinical trials, which may compromise the scientific integrity of data and may raise concerns for patient safety. Moreover, there is an unprecedented operational burden on trials’ conduction as there is limited access to trial activities and investigations of novel therapies or interventions for various diseases, particularly involving vulnerable populations ,13. The Randomised clinical trials (RCTs) provide the highest quality of evidence for developing newer therapies or interventions to treat challenging diseases, thus improving the quality of life (QoL). Until March 2020, the ClinicalTrials.gov, an international clinical trial registry, documented a total of 262,366 ongoing RCTs, most of which are registered for investigating a drug or biological intervention n=146,420), followed by trials on behavioural studies  and based on surgical/device interventions  [6,420, foFor universities and the pharmaceutical industry, useful and timely management of clinical trial operations is crucial due to the substantial financial investment . Furthermore, large trial centres usually run multiple trials at any given point of time, enrolling thousands of patients at various sites worldwide. Therefore, this pandemic disrupts operational planning, decision-making, and creates operational gaps that can significantly affect the financial cost and patient safety with interruption of treatment leading to potential harm.To date, many institutions and organisations have already paused their clinical trials. There are several critical challenges in the current pandemic which include: (a) limited accessibility of clinics mainly for essential or critical visits, (b) there is difficulty in recruiting homebound patients, or others are also reluctant to visit clinics, (c) currently trial operations may expose the staff or patients to the risk of acquiring the infection, (d) continuation of the trial may lead to a high drop-out rate, (e) inability to meet logistical trial obligations by sponsors as well as contractors , and (f) deviation from the study timelines may affect data integrity due to delayed assessment and monitoring.Notably, COVID-19 prevents several institutions from continuing their previously existing trial activities. Disruption of trial programmes has resource, ethical, and care implications. In order to continue the trial operations, it is necessary to follow the regulatory guidelines and regularly review the current infection trend, oversight of lockdown policies and travel restrictions, future prediction of COVID-19 and understanding key risk indicators. Based on these trends, appropriate recommendations and policies should be followed at the trial sites, and risk-based decision making should use evidence from both epidemiologists and health policy-makers. These decisions regarding trials may consider changing study sites, extending programmes, and amendment of planned trial closure, monitoring and screening activities, site access, and, where appropriate, application of machine learning-driven forecasting models. As these changes require time and resources, so it is crucial to have robust risk/issue trackers together with the better symbiosis between operational- and strategic-level decision-making.Notably, the concept of telemedicine exists for the past two decades. However, its integration into clinical practice has specific administrative and bureaucratic challenges, mainly involving the cost of implementation and reimbursement that is far beyond the conceptualization or technological shortfalls . HoweverWe suggest that adopting telemedicine in the clinical research settings, assessing the global impact of decisions, developing contingency plans, providing additional financial support, greater flexibility and understanding may be useful to help solve this problem. Clinical trials necessitate colossal investment in terms of finance, logistics and other resources (including participants’ time). Similar to the previous pandemics, the impact of the COVID-19 on clinical trials will hopefully be short-lived; meanwhile, to maximize the public health benefits of existing trials, we need creativity and persistence in these unprecedented times.Participants must be self-isolated, and research staff needs to work remotely to avoid direct contact. A framework for detection, recording, assessment, and reporting of all procedural deviations is required, and the appropriate board should be in place to monitor the progress of the clinical trial site(s). All protocol deviations that could lead to severe risk for the patient must be reported. The trial must always be conducted in-line with the Good Clinical Practices (GCP) guidelines.Monitoring activities should be reassessed to ensure the safety of study participants. Besides, some processes may have to be modified, such as electronic alternatives for signatures that may have to be considered. As administrative personnel may not be able to meet study teams or answer direct calls personally, the use of teleconferencing and voice mails during the pandemic will be useful.Isolation/quarantine criteria can lead to deviations intended to remove a research participant’s immediate apparent threats. For example, if the participant is unable to attend the treatment appointment, and if the only re-scheduling date available is outside of the study visit period duration, no research protocol or doses should be missed. In this case, a missed dose or procedure for the maintenance of the security and welfare of the subject should not cause any potential harm. So, the health and safety of the participants in the study should be maintained and prioritized. Also, participants must be aware of the risks/changes associated with the study protocol that could affect their well-being. All participants affected by a COVID19-related study disruption should be documented by a unique participant identifier and description of how the individual’s participation was altered. In the case of a visit to the clinical trial site, the researchers may have to assess whether alternate safety assessment measures are feasible if participants are unable to come to research sites as set out in the study protocol.Alternative methods may include telephonic conversation or the use of telemedicine for virtual visits or alternative sites for care/collecting study data. Alternative locations may need to be considered for imaging and laboratory testing as per study protocol. In the case of alternative monitoring, careful documentation will be required to obtain the reason, how information has been collected, what data has been collected, who has provided the information; how the information sources have been verified and why. No amendments to the study protocol are required.Researchers are encouraged to consider whether they should hold all the study procedures or part of it during the pandemic. This can refer to all procedures or part of a study. Maybe it is necessary to hold registration, study visits, collect or analysis of data, etc. It is understood that the cessation of the treatment of a patient might not be unsafe or may seriously affect project performance. So, the decision to place a hold on a particular study needs to be made in consultation with the institutional governing body to safeguard the study participants’ and departments’ needs. , 19Finally, in order to efficiently address the anticipated circumstances by data monitoring committees during the pandemic, special provisions should be placed. For instance, data assessment regarding the benefits and risks of interventions for COVID-19 requires faster communication during health emergencies . This coClinical trials are of paramount importance for the advancement and development of novel treatment interventions. The ultimate goal of such trials and studies is to ensure the best quality patient care with the highest and most favourable outcome, whilst decreasing the cost and suffering of (a burden to) the recipient. Such endeavours are vital to the medical community. For the functioning of a robust medical system wherein discoveries, drugs and technology can be applied to the system from the top tier and eventually diffuse out to primary and satellite care centres to ensure the best care for all.Historically, clinical trials have usually been a laborious, expensive, and difficult task to undertake under normal circumstances; however, trials under the unprecedented COVID-19 conditions have indeed suffered its consequences.The often complicated design and intricate nature of clinical trials mean the likelihood of derailment is high. A cohort of multiple external factors has to be controlled and maintained to ensure the sanctity of the data as well as the integrity of the trial. All the criteria mentioned above are nullified by a pandemic such as COVID-19.For the commencement of a new clinical trial or even the maintenance of an existing trial, funding is a priority in order to drive it to completion. COVID-19 severely impeded funding of non-COVID-19 related trials as the acquisition of a cure for the virus took centre stage for international funding bodies. Over and above the funding issue, the need to tightly monitor trial participants and subsequently ensure their safety through such a pandemic proved detrimental to many ongoing studies, or those recently started. The statistics show a sharp decline in the enrolment of new members for trials.Clinical trials did not only compete for funding under COVID-19 conditions but simultaneously had to cope with lockdown regulations that made accessibility of the study participants virtually impossible. The fact that data from the ongoing studies will be incomplete or will have gaps could lead to either shutting down the trial, a severe delay , or the need to re-start the trial. The ramifications of COVID-19 causing such a massive international dearth in the commencement of new trials and the continuation of non-COVID-19 trials will not be evident in the immediate future. However, the impact thereof will become evident as the time lost will add up to years lost in research in the broader spectrum.COVID-19 and its deleterious effects on trials call for a possible re-structuring of how trials are conducted. A feasible, practical and scientifically sound alternative to conventional trial studies is needed. The alternative will need to ensure scientific integrity but will also need to balance the needs of the parties involved to overcome various difficulties due to external factors. A solution to trials and research as well as time and funding lost during lockdown due to COVID-19 must be addressed, ensuring scientific breakthroughs and the development of new technology is not hindered or delayed. The lives of millions of patients are dependent on the continual clinical breakthroughs and discoveries which are produced by these trials.The COVID-19 pandemic has resulted in a series of public health policies that have crippled the healthcare systems of many countries. This situation hugely impacted the study participants, care providers, researchers, trial sponsors, and research organizations concerning clinical trials. This pandemic has a substantial impact on the trial sites as they experience difficulty in the continuation of trial activities which eventually hampers the progress of the trial and delays study timelines. Most sites are struggling due to delayed subject enrollment, shortfalls in monitoring, and risks of compromised data integrity, and this situation has negatively impacted the initiation of future trials as well. Researchers are also concerned regarding the delay or cancellations of trials in this situation, which will have a financial impact on research organizations and human resources. According to one survey, about two-thirds of the respondents have stopped or will soon halt subject enrollment in ongoing clinical trials, one-third halted randomization, and fifty percent of respondents are delaying or planning to delay the studies. Therefore, using dynamic, proactive strategies and a framework for decision-making and risk assessment is needed to overcome these challenges in conducting clinical trials. Adopting new approaches and understanding the key risk indicators will help managers support trial sites with flexibility and ingenuity. For instance, switching patient site visits to new-trial virtualization, and telemedicine to interact with patients will help manage current clinical trials also beneficial for the post-pandemic era.


Interphotoreceptor retinoid-binding protein (IRBP), also known as retinol binding protein 3 (RBP3), is a lipophilic glycoprotein specifically secreted by photoreceptors. Enriched in the interphotoreceptor matrix (IPM) and recycled by the retinal pigment epithelium (RPE), IRBP is essential for the vision of all vertebrates as it facilitates the transfer of retinoids in the visual cycle. It also helps to transport lipids between the RPE and photoreceptors. The thiol-dependent antioxidant activity of IRBP maintains the delicate redox balance in the normal retina. Thus, its dysfunction is suspected to play a role in many retinal diseases. We have reviewed here the latest research on IRBP in both retinal health and disease, including the function and regulation of IRBP under retinal stress in both animal models and the human retina. We have also explored the therapeutic potential of targeting IRBP in retinal diseases. Although some technical barriers remain, it is possible that manipulating the expression of IRBP in the retina will rescue or prevent photoreceptor degeneration in many retinal diseases. Interphotoreceptor retinoid-binding protein (IRBP), also known as retinol binding protein 3 (RBP3), was first discovered in soluble proteins extracted from the bovine interphotoreceptor matrix (IPM), which is located between the neural retina and the retinal pigment epithelium (RPE)  and orthodenticle homolog 2 (OTX2), two essential transcriptional factors in photoreceptors  accumulation, mitochondrial dysfunction and cell death   in IRBP were identified in four children diagnosed with retinal dystrophy and myopia  of Downregulation of IRBP also caused the accumulation of the retinal 'waste product' lipofuscin, which may increase the risk of oxidative damage to the RPE and photoreceptors  Ambros, . More thhttp://www.targetscan.org/vert_72/)  and transcription activator-like effector nucleases (TALENs), the third generation, clustered regularly interspaced short palindromic repeats (CRISPR) technique has recently risen (Doudna and Charpentier, IRBP is required to maintain the normal functions of the retina, and its downregulation is a common phenomenon at the early stages of photoreceptor degeneration. Although it may be an initial defensive response to retinal stress, the suppression of IRBP is harmful to the health of the photoreceptors in the long term . The cloTZ and LZ conceived the perspective of the work. SZ, TZ, YC, LH, and MP searched and summarized the literature. SZ and TZ drafted the manuscript. TZ, RA-B, LZ, and RN designed the figure. MG, NF, RN, FZ, and MM critically revised the article. All authors commented on and contributed to the final 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.