One of the primary causes of chemotherapy failure is the occurrence of cancer multidrug resistance (MDR). Uncontrolled growth of resistant tumor cells leads to metastasis and recurrence, associated with high mortalities. Ferrocifens have been shown to possess anticancer properties via an original mechanism dependent on redox properties and generation of active metabolites that can cause disruption of cell metabolism. However, these molecules are highly insoluble in water, requiring a formulation stage before being administered in vivo. Lipid nanocapsules (LNCs) have already demonstrated their ability to successfully encapsulate various hydrophobic therapeutic agents, such as ferrocifens, and offer the option of surface modification, making it possible to adapt the pharmacological behavior of the nanocarrier. The aim of this review is to give, for the first time, an overview of the in vitro and in vivo studies performed with ferrocifen-loaded LNCs on several MDR cancers.Resistance of cancer cells to current chemotherapeutic drugs has obliged the scientific community to seek innovative compounds. Ferrocifens, lipophilic organometallic compounds composed of a tamoxifen scaffold covalently bound to a ferrocene moiety, have shown very interesting antiproliferative, cytotoxic and immunologic effects. The formation of ferrocenyl quinone methide plays a crucial role in the multifaceted activity of ferrocifens. Lipid nanocapsules (LNCs), meanwhile, are nanoparticles obtained by a free organic solvent process. LNCs consist of an oily core surrounded by amphiphilic surfactants and are perfectly adapted to encapsulate these hydrophobic compounds. The different in vitro and in vivo experiments performed with this ferrocifen-loaded nanocarrier have revealed promising results in several multidrug-resistant cancer cell lines such as glioblastoma, breast cancer and metastatic melanoma, alone or in combination with other therapies. This review provides an exhaustive summary of the use of ferrocifen-loaded LNCs as a promising nanomedicine, outlining the ferrocifen mechanisms of action on cancer cells, the nanocarrier formulation process and the in vivo results obtained over the last two decades. The field of oncology is predominant and has been of increasing interest since the anticancer nanomedicine Doxilapproval ,2,3. Oveapproval . The maiapproval ,5. Tamoxapproval . Paclitaapproval . Howeverapproval .In the late 1980s, Jaouen et al. synthesized a new class of bio-organometallic molecules . The ideThree generations of nanoparticles (NPs) can be differentiated. In the 1970s, the first nanomedicines were biodegradable but accumulated mainly in the liver. The second generation is characterized by the surface modification with hydrophilic molecules, mainly polyethylene glycol (PEG), in order to confer stealth properties and to passively accumulate in solid tumor tissues via the so-called enhanced permeation and retention (EPR) effect ,15. HoweThe aim of this review is to give, for the first time, an exhaustive overview on the in vitro and in vivo studies performed with ferrocifen-loaded LNCs. Firstly, ferrocifen mechanisms of action at the cellular level will be detailed in relation to potential anti-MDR activity. Then, the specificities of LNCs will be described, especially through their potential as ferrocifen nanocarriers, and the in vivo experiments on MDR cancers such as glioblastoma, metastatic melanoma and breast cancer will be reviewed.Ferrocifen compounds were synthesized for the first time in 1996 by Jaouen et al. ,34. The 1) or to change the functional group R2 has allowed the synthesis of more than three hundred complexes over the last two decades, some of them showing anticancer properties on a large panel of MDR cancer cell lines including glioblastoma, metastatic melanoma, breast cancer and leukemia [The ease of ferrocene functionalization and the ability to shape the overall structure by adding a basic side chain  can only occur if the ferrocenyl group is located on carbon 2 of the but-1-ene group and if the phenol is on carbon 1 . This co50  and of growth inhibition concentration GI50 (concentration inhibiting 50% of cell proliferation), found for the different ferrocifens: all the different values are in the low or submicromolar range. This can be explained by the multiple pathways of activity, as discussed below.Early studies proposed some possible tumor cell mechanisms due to the well-known antiestrogenic effect of ferrocifens (cytostatic effect) ,53. Inte2)3NMe2 in P15 (group R1 in 2)2N(CH3)2. This can be explained by the fact that the lone pair of the nitrogen needs to interact by hydrogen bonding with the Asp351 of the estrogen receptor to provide the right position in the ER pocket, leading to the correct ER conformation [The antiestrogenic proliferative effect of the ferrocifens has been well-discussed in the literature. With their molecular structures, they can bind to the estrogen receptor, filling the hydrophobic pocket  and, by ormation . This isormation ,52,54; iThe cytotoxic effect of the ferrocifens seems to be related to the unique redox properties of ferrocene, able to induce a reversible oxidation of Fe(II) to Fe(III) (Fenton reaction), followed by the production of ROS known for their bioreactivity ,65, and 50 = 0.15 µM) [2)3N(CH3)2, a lipophilic cation in the cancer cell environment, allowing a preferential localization in mitochondria. According to Scalcon et al., this overexpression of oxidized thioredoxin in mitochondria led to an increase in ROS and the release of cytochrome c, which can activate the caspase-cascade system, enzymes responsible for apoptosis [As mentioned above, the formation of QM has been suggested as one of the principal causes of the cytotoxic effect ,38,68. I0.15 µM) . The hig0.15 µM) ,52. Anot0.15 µM) ,67,70. Ipoptosis ,72.Thus, ferrocifens could be seen as promising anticancer compounds as they provide a variety of mechanisms of action, depending on the cell line treated, the concentration used and their molecular structure. For example, by forming QM, ferrocifens behave as ROS-producing compounds by increasing the ROS level inside an oxidative stress environment. Moreover, it may be possible to consider ferrocifen as a prodrug: the corresponding QM inhibits cytosolic and mitochondrial TrxR more efficiently than the parent compound. However, in order to further analyze the efficacy of these molecules and to perform in vivo studies, ferrocifens need to be formulated in nanocarriers. Indeed, due to their high lipophilicity, they cannot be injected as a solution into the circulatory system and lipid nanocapsules could represent one potential answer to this.Nanometric colloidal systems, as drug carriers, are dispersed systems, often oil in water dispersions, in which solid or liquid phases are in suspension in a fluid medium. The main process to obtain these systems is to use emulsification or nanoprecipitation methods. However, one drawback of these methods is the use of organic solvents, required to solubilize the excipients, mainly lipids or polymers . Moreove® WR 1349), nonionic hydrophilic polyethylene glycol (15)-hydroxystearate surfactant (Solutol® HS 15) and, as an amphiphilic cosurfactant, phosphatidylcholine from soybean (Lipoid®). The last principal component is the aqueous phase, MiliQ® water, containing a fixed amount of sodium chloride (NaCl) to make the suspension injectable [w/w) of polyethylene glycol (15)-hydroxystearate; 35–80% (w/w) of water; 10–25% (w/w) of caprylic/capric triglycerides. It should be noted that each constituent has an influence on LNC formulation and stability [LNCs are composed of three main parts: an oily phase, an aqueous phase and nonionic surfactants. The oily phase is composed of caprylic/capric triglycerides -hydroxystearate is oriented towards the water phase c 24,73,,73,77.2000) coating in the design of long-circulating LNCs [1500 stearate after an intravenous injection in a murine model. The results obtained revealed that this LNC modification allowed a very weak complement consumption and an increase in the blood circulation half-time, by reducing the protein adsorption, and hence the opsonization, due to an efficient steric hindrance [2000-coated LNCs improved the biodistribution in a murine model by extending the blood circulation time associated with good structural stability over several hours, after intravenous injection [The principal drawback of NPs for drug delivery is the fact that these nanocarriers are recognized by the mononuclear phagocyte system (MPS) as foreign bodies. Indeed, after a systemic injection, the clearance of nanocarriers from the blood can be very rapid, due to the macrophages of the MPS, particularly Kupffer cells in the liver, and the complement system. Plasma protein such as C3b molecules (named opsonins), mostly present in blood, can bind to the surface of the NPs due to weak forces such as electrostatic and hydrophobic interactions, forming a protein corona (opsonization). Then, opsonins are recognized by macrophages, leading to the phagocytosis of the NPs ,79,80,81ing LNCs ,84,85,86ing LNCs . Due to indrance . These rnjection ,83,88,892000 functionalized with reactive-sulfhydryl maleimide groups  to perform an active targeting strategy via the covalent grafting of a homing moiety such as monoclonal antibody to the maleimide function [Moreover, the use of DSPE-PEG can be of interest for covalent functionalization, offering the possibility of shaping the terminal group of the molecule . For exafunction . In anotfunction . Moreovefunction . Finallyfunction ,94. Indefunction , to funcfunction . Due to function .Most of the ferrocifen compounds synthesized in recent decades have shown a promising efficacy on the National Cancer Institute (NCI) panel of 60 cancer cell lines , as presented in 50 values were not affected by the encapsulation of ferrocifens into LNCs, whatever the formulation used. Indeed, Lainé et al. showed that the IC50 of free P15 on MDA-MB-231 breast cancer cells was 2 µM. The same value was found after LNC encapsulation [50 on U87 MG glioma cells free or encapsulated into LNCs, with even a better efficacy after NFL peptide adsorption on P53 LNCs [50 [50, 1 µM , DP1-loaded LNCs blocked cells in the G0/G1 phase and led to apoptosis. This was confirmed by the counting of the apoptotic cells: 15% of apoptotic cells at 0.5 µM of DP1 compared to 5% at 0.1 µM . Using a50 of P5 on glioma 9L cells was about 0.5 µM. This phenomenon was explained by the fact that P5 could take advantage of the microenvironment of the cancerous cell, rich in ROS and leading to the corresponding QM, whereas the high amount of antioxidant proteins such as glutathione in astrocytes allowed an efficient scavenging of ROS. Another explanation advanced by the authors would be due to the easier accessibility of ferrocifens to the intracellular target during cell division. The same result was observed with conventional loaded LNCs, confirming the theory that the low toxicity observed in healthy cells is due to the mechanism of action of these organometallic compounds and not to an active targeting strategy. However, in some studies, the surface modification of the LNCs with active ligands  was studied, either because they did not contain ferrocifens [Finally, while ferrocifens demonstrated outstanding effects on various cancer cell lines, it is important to note that these compounds resulted in little or no toxicity on healthy cells, such as melanocytes and astrocytes, even after encapsulation into LNCs ,39. Inderocifens  or becaurocifens .All the in vivo studies carried out on ferrocifen-loaded lipid nanocapsules (LNCs) are summarized in Malignant brain tumors are among the most severe cancers due to their poor diagnosis and life expectancy. Glioblastoma is the most common and aggressive type of primary malignant brain tumor, resulting in the assignment of grade IV, the highest grade in the World Health Organization classification . Gliobla3 for untreated rats to 300 mm3 for treated rats after 21 days  phenomenon and hepatotoxicity . Even if 21 days c, showinBreast cancer is one of the most common cancers, corresponding to 15% of all cancer deaths among women. Metastasis to other organs such as the liver, lung, brain and bones, is frequent and accounts for the majority of deaths from this cancer ,108. Hop50 = 1 µM for DP1; IC50 = 3 µM for P5 and IC50 > 100 µM for DITC) [Malignant melanoma is the leading cause of death from skin cancer with a 5-year survival rate of less than 10%. Normally, surgery is an adequate treatment when the tumor is diagnosed early enough. A chemotherapy treatment with dacarbazine (DTIC) is additionally applied, with a low response rate . There ior DITC) .Moreover, as previously mentioned for 9L glioma cells , this st8+ T lymphocytes after treatment of stealth P722-LNCs, leading to immunogenic cell death [Finally, it was shown for the first time, in another recent study, that some ferrocifens could induce a new therapeutic pathway in resistant melanoma cancer. Indeed, as previously described, P722-loaded LNCs were used on an orthotopic murine model of human B16F10 melanoma. In addition to an induction of apoptosis via the intrinsic pathway, the results revealed a significant increase in activated CDll death .As previously described, glioblastoma remains difficult to treat despite improvements in chemotherapy and radiotherapy ,112,113.Several studies highlighted the idea that overexpression of Bcl-2 could be responsible for the chemoresistance of melanoma ,115. BclAnother potent strategy to target cancer cells with NPs is to use mesenchymal stromal cells (MSCs), such as marrow-isolated adult multilineage inducible (MIAMI) cells, a subpopulation of MSCs. In glioblastoma studies, it has been highlighted that MIAMI cells were mainly localized at the border between tumor cells and healthy brain cells. For this application, LNCs were efficiently internalized into MIAMI cells without any negative effects on cell viability and differentiation, allowing their migration towards U87MG glioma cells. However, such modified MIAMI cells did not modify the survival of U87MG-bearing mice ,121,122.Since their first synthesis at the end of the 1990s, ferrocifens, molecules of the organometallic family of compounds, have been under constant investigation to understand their mechanisms of action. They have shown remarkable in vitro antiproliferative and cytotoxic effects on various cancer cell lines. Their mechanisms of action are different from those of the usual chemotherapeutic compounds , well-known DNA-damaging or microtubule-stabilizing agents and SERMs. Indeed, ferrocifens are molecules endowed with multiple biological properties, including anticancer activity, that induce inhibition of cancer cell growth through apoptosis and/or senescence according to the conditions previously described . Additionally, by taking advantage of the microenvironment of the tumor (acid pH and oxidative stress), these organometallic compounds might overcome the resistance of MDR cancer to apoptosis. Moreover, ferrocenyl quinone methide, which has selective electrophilic reactivity, can interact with thioredoxin reductase and so inhibit its activity, leading to the production of ROS. These promising in vitro results have been confirmed widely in in vivo experiments by using LNCs as nanocarriers to encapsulate the highly lipophilic ferrocifens. Whether on ectopic or orthotopic models, whatever the cancer cell lines tested—glioblastoma, breast cancer, metastatic melanoma, all well-known for their chemoresistance to current therapy—in vivo treatment by ferrocifen LNCs revealed, in most cases, an increased median survival rate in animal models. The rationale for combining therapies using drugs or other strategies  operating via different mechanisms and/or acting synergistically, and thus decreasing the likelihood that resistant cancer cells will develop, was also successfully tested with ferrocifen LNCs.However, even if there is evidence for the effictiveness of these nanomedicines, it is important to keep a critical eye on the results obtained as they can vary depending on the selected model—ectopic or orthotopic. For example, it is well-known that orthotopic tumor models develop a tumor microenvironment closer to the reality than the ectopic tumor models . Moreove


Sphingosine-1-phosphate (S1P) receptors are extensively used in the treatment of multiple sclerosis (MS). However, the optimal therapeutic role of S1P in MS patients has still remained elusive. This network meta-analysis (NMA) systematically evaluated the efficacy and acceptability of S1P receptors, as disease-modifying drugs, in the treatment of patients with MS, so as to find out the most appropriate therapeutic strategy and provide a reliable basis for the prescription of S1P drugs for patients with MS.ClinicalTrials.gov databases. The primary outcome in this study was the treatment efficacy for the S1P receptor for MS patients, in terms of decrease in annualized relapse rate. The secondary outcomes were adverse events leading to discontinuation of a study, such as an unfavorable or unintended sign/symptom. Outcomes were appraised using a random effects model expressed as standardized mean differences (SMDs) and risk ratios (RRs) with 95% confidence intervals (CIs), respectively, and were ranked using surface under the cumulative ranking curve (SUCRA) probabilities for hierarchical clustering of interventions.We conducted a systematic review and NMA to compare the efficacy and acceptability of S1P receptors for treating MS patients. Randomized controlled trials (RCTs), which were published until May 2020, were retrieved from the PubMed, Cochrane Library, Embase, and A total of 13 RCTS were included, which enrolled 10,554 patients. The results of NMA showed that Fingolimod, Laquinimod, Siponimod, Ozanimod, Amiselimod, and Ponesimod were superior to placebo in terms of reducing the annualized relapse rate of MS patients. Regarding efficacy, the best and worst treatments were Amiselimod  and placebo (SUCRA 90.5%), respectively. As for acceptability, the best and worst interventions were Ozanimod  and Ponesimod , respectively. The comparison-adjusted funnel plots of annualized relapse rate and side effects in the included studies revealed that there was no significant funnel plot asymmetryThis NMA indicated that Amiselimod (0.4 mg) is the most effective treatment strategy as a S1P receptor for MS patients. However, the abovementioned findings need to be further confirmed in the next researches. Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS), in which inflammation, demyelination, and axonal loss occur in even early stages of the disease. Nevertheless, in the majority of patients, MS is characterized by recurrent relapses, followed by complete or partial recovery. Recurrent attacks and disease progression eventually lead to an irreversible neurological damage . TherefoA network meta-analysis (NMA) can be conducted to compare multiple treatments that may not have been compared directly in head-to-head clinical trials. The appropriate use of NMAs can lead to enhanced decision-making in situations where head-to-head clinical trials do not exist; however, decision makers need to be aware of the potential challenges that can arise if NMAs are conducted that do not adequately adjust for cross-trial heterogeneity The present NMA systematically evaluated the efficacy and acceptability of S1P receptors, as disease-modifying drugs, in the treatment of patients with MS, so as to find out the most appropriate therapeutic strategy and provide a reliable basis for the prescription of S1P drugs for patients with MS.ClinicalTrials.gov databases. The titles and abstracts of studies retrieved from the search process were read, and we thoroughly reviewed the full texts of relevant articles to determine whether the retrieved studies were eligible to be included in this NMA. Disagreements were resolved by a third reviewer or consensus-based discussion. The search process was carried out using the following search items: [(multiple sclerosis) OR (MS)] AND [(s1p receptor)OR (s1pr) OR (s1pr1) OR (s1pr2) OR (s1pr3) OR (s1pr4) OR (s1pr5)], without any language restrictions. Regarding eligibility, only parallel RCTs were selected, whereas crossover trials, single-arm trials, case reports, and conference papers were excluded. The study population included MS patients who were treated with S1P receptors. However, in the process of literature search, we found that AhR receptor, e.g., Laquinimod, had also been used as a substitute for Fingomod in the treatment of MS. Therefore, we included MS patients who were treated with Laquinimod as a secondary observational indicator to compare the efficacy and acceptability of S1P receptor with AhR receptor.NMA, in the context of a systematic review, is a meta-analysis, in which multiple treatments  are compared using both direct comparisons of interventions within randomized controlled trials (RCTs) and indirect comparisons across trials based on a common comparator . RCTs, wTwo reviewers independently classified the therapies, extracted the key study parameters through a standardized data abstraction form, and assessed the quality of trials and the risk of bias according to the Cochrane Collaboration’s tool . DisagreThe primary outcome in this study was the treatment efficacy for S1P receptor for MS patients, in terms of decreased annualized relapse rate (ARR). The secondary outcomes were adverse events leading to discontinuation of a study, such as an unfavorable or unintended sign/symptom.The NMA was conducted using Stata 15.0 software . When dichotomy was applied to outcome measures, risk ratio (RR), and 95% confidence interval (CI) of each outcome indicator were calculated. If the CI does not contain the null hypothesis value, the results are statistically significant. Standardized mean difference (SMD) and 95% CI of each outcome indicator were calculated when the outcome variable was continuous. In the presence of three-arm or more trials, the two arms of all possible combinations were first broken, and a network of evidence for making comparison between various treatments was then established. To do this, we first completed a network of evidence for each outcome measure, in which the size of each node represented the total number of subjects in each intervention, and thickness of each line indicated the number of studies that compared the two interventions. In case of the existence of a network diagram, which is a graphical depiction of the structure of a network of interventions, calculate inconsistency factor (IF), and its 95% CI to evaluate the consistency of each closed-loop. The lower limit of 95% CI is equal to 1, indicating good consistency; otherwise, the closed loop is considered to have obvious inconsistencies. In order to facilitate the process of the interpretation of odds ratio (ORs), the probability of each intervention was computed as the safest or the most satisfactory treatment method of the Bayesian approach based on probability values, and thus summarized as surface under the cumulative ranking curve (SUCRA). A larger SUCRA value symbolized a better rank of intervention. The smaller the SUCRA is, the better the treatment measures will be. Cluster analysis (CA), a multivariate tool used to organize a set of multivariate data  into groups called clusters, was employed to find out the most appropriate intervention measures. A comparison-adjusted funnel plot was developed to indicate whether publication bias would be existed in the ARR and the rate of discontinue due to adverse events. For the evaluation of inconsistency between the direct and indirect comparisons conducted in the eligible studies, we used the design-by-treatment-interaction model, loop-specific approach, and node-splitting model .n=2619; Laquinimod, n=1090; Siponimod, n=1148; Ozanimod, n=872; Amiselimod, n=312; and Ponesimod, n=337). Figure Figure Table The side effects included atrioventricular block, leukoencephalopathy, elevated level of alanine aminotransferase (ALT), dyspnea, and infection. Treatment was terminated as critical side effects were reported in each study.The results of treatment efficacy and acceptability are presented in Table Furthermore, Fig. We further evaluated the inconsistency of these outcomes using the design-by-treatment, loop-specific approach, and node-splitting model, and found no evidence of statistical inconsistencies Fig. . It shouMS is categorized as a rare disease with an estimated 30,000 MS patients in China. Due to the pathological changes of the autoimmune system, the nerve myelin sheath is damaged and peeled, resulting in the impairment of the spinal cord, brain, and optic nerve’s functions. It is an inflammatory demyelinating CNS disease frequently appearing in young adulthood. Supported by an experimental evidence, MS is generally considered a predominantly T cell-mediated autoimmune disease. A typical early disease course is characterized by clinical relapses followed by symptomatic improvement or remissions with an indolent accumulation of symptoms later in life, likely in part due to a component of neurodegeneration as well as ongoing chronic inflammation .S1PR1 is located on lymphocytes, neural cells, endothelial cells, smooth muscle cells, and atrial myocytes, as well as in the atrioventricular node and the conduction system. Functions include the egress of lymphocytes from lymph nodes, neuron migration and function, endothelial permeability, vasculature formation, in addition to the decreased heart conduction. S1PR2 is located in the CNS and on endothelial and smooth muscle cells, influencing hearing and balance function, as well as endothelial permeability and vascular tone. S1PR3 is located on neural cells, atrioventricular node, and the conduction system, as well as on smooth muscle cells. Its functions comprise neural cell migration and function, slowed cardiac conduction, and endothelial permeability. S1PR4 is located solely on lymphocytes, influencing lymphoid tissue expression, as well as modulation of dendritic cells and TH17 cells. S1PR5 is located in the CNS. S1P binds to G-protein-coupled receptors (S1P1–S1P5) to modulate a wide range of physiological systems .To date, several multi-center, double-blinded, randomized, controlled trials have been carried out on S1P receptor. However, the lack of head-to-head comparisons has made it impossible to compare the efficacy and acceptability of s1PR. In the present NMA, the efficacy and acceptability of the interventions were ranked by indirect comparison among studies with an insufficient evidence: a total of 13 RCTs and a total of 10,554 patients were herein included. Compared with placebo, S1P receptors, such as Fingolimod, Siponimod, Ozanimod, Amiselimod, Ponesimod, and Laquinimod could significantly reduce the ARR and disability progression. According to the results of this NMA, Amiselimod (40 mg) possessed the highest efficacy, followed by Ponesimod (40 mg), and Laquinimod had the worst efficacy. In terms of safety, Fingolimod (1.25 mg) and Ponesimod (40 mg) showed a great number of dropouts due to severe adverse reactions compared with placebo, while other drugs did not cause significant adverse reactions compared with placebo. Ozanimod (1 mg) is a drug with the lowest risk of serious adverse reactions, leading to trial discontinuation. It was revealed that Amiselimod (40 mg) possessed the highest treatment efficacy with the least side effects.in vitro) more slowly than Fingolimod, because it was thought that its effect on the heart rate of humans may be ameliorated by a gradual increase in concentration of its active metabolite in the heart following administration. Amiselimod-P could result in an approximately five-fold weaker activation of GIRK channels in human primary atrial myocytes compared with Fingolimod-P. Because the S1P1 agonist activity of Amiselimod-P was reported similar to that of Fingolimod-P, the potential for bradycardia caused by Amiselimod-P is likely to be less than that for Fingolimod-P, owing to weaker activation of GIRK channels [Amiselimod was designed as a prodrug S1P receptor modulator lacking S1P3 agonism to avoid bradycardia with the first dose. It is converted to its active metabolite, and it functions as a highly selective S1P1 functional antagonist without S1P3 activity. In addition to its lack of S1P3 agonist activity, it was designed to be converted to the active metabolite in human cells  indicated a higher concentration of Fingolimod in the CNS compared with peripheral compartment, accumulating in white matter and myelin sheaths plays a significant role in modulation of several processes in the CNS, including maturation, proliferation, and migration of neuronal cells that interact and balance brain damage and repair. Although Fingolimod primarily binds to S1PR1, and the other S1P receptors are also affected by Fingolimod therapy. Based on the natural distribution pattern of the S1P-receptor subtypes, especially on atrial myocytes, the risk of cardiac events has notably increased .Ozanimod does not require phosphorylation for activation and it induces a rapid, dose-dependent, and reversible selective reduction. It is an agonist of the S1PR1 and S1PR5 with a 27-fold selectivity for S1PR1 over S1PR5. Its effects can be further mediated by S1PR1 receptor internalization and subsequent ubiquitin-proteasome-dependent degradation, thereby preventing receptor reinstallation in the cellular membrane. Ozanimod binding to S1PR5 can activate specific cells in the CNS, promote myelin regeneration, and prevent synaptic defects, ultimately preventing nerve damage. Ozanimod possesses an excellent advantage in terms of reducing the ARR due to the combination of the two mechanisms “damage reduction + repair enhancement” .Siponimod influences both peripheral B and T cells with pronounced effect on CD4 T cells compared with CD8 T cells, as well as a preferential decline in CD4 naive cells and CD4 central memory T cells  with less effect on CD4 peripheral effector memory T cells . Siponimod decreases oligodendocyte and axonal loss, suggesting that it might be able to protect axons during both the acute and the chronic demyelinating phases of MS. There was no significant effect on remyelination . A meta-in vitro, Ponesimod is at least 10-fold more potent on the S1P1 receptor than on other S1P receptor subtypes. Lymphocytes migrate from the lymph node into blood following an S1P gradient that is maintained by the high levels of S1P in the blood and lymph, which far exceed those at tissues. Binding of Ponesimod to the S1P1 receptor results in rapid and efficient receptor internalization, degradation, and functional antagonism, thereby causing lymphocyte sequestration in the lymph nodes [Ponesimod, an iminothiazolidinone derivative, is a reversible, orally active, selective S1P1 modulator. In contrast to Fingolimod, which is a structural analogue of sphingosine, Ponesimod is selective for S1P1; ph nodes .The present NMA contains a number of limitations. First, the majority of the included studies did not explicitly report random methods, allocation, and concealment schemes and blind methods, and there was possibility likelihood of selective bias, implementation bias, and measurement bias. Second, the research outcome indicator reports do not have a unified standard. For instance, when ARR was reported, the affiliated data were partly expressed as mean ± 95% CI, and some as mean ± standard error of the mean (SEM). Third, in the presence of some factors, e.g., China’s economic and social conditions, several RCTs on administration of drugs have been conducted in China, while further multi-center, long-term, and double-blinded studies are warranted. It should be noted that the majority of studies included in this NMA were conducted in European counties and in the USA, and there may be a significant difference in treatment efficacy between the European and Asian populations.In summary, S1P receptors are effective in terms of reducing the ARR in patients with MS. Comparing the efficacy and safety of some therapies for MS patients showed that Amiselimod (40 mg) possesses a promising efficacy in terms of reducing the ARR and a low adverse reaction rate. Comparably, Fingolimod possesses satisfactory therapeutic effects, while it has a higher adverse reaction rate. However, the abovementioned conclusions need to be further confirmed in the next researches.


To systematically study the mechanism of cordyceps cicadae in the treatment of diabetic nephropathy (DN) with the method of network pharmacology and molecular docking analysis, so as to provide theoretical basis for the development of new drugs for the treatment of DN.  TCMSP, Symmap, PubChem, PubMed, and CTD database were used to predict and screen the active components and therapeutic targets for DN. The network of active components and targets was drawn by Cytoscape 3.6.0, the protein-protein interaction (PPI) was analyzed by the STRING database, and the DAVID database was used for the enrichment analysis of intersection targets. Molecular docking studies were finished by Discovery Studio 3.5.  A total of 36 active compounds, including myriocin, guanosine, and inosine, and 378 potential targets of cordyceps cicadae were obtained. PPI network analysis showed that AKT1, MAPK8, and TP53 and other targets were related to both cordyceps cicadae and DN. GO and KEGG pathway analysis showed that these targets were mostly involved in R-HSA-450341, 157.14-3-3 cell cycle, and PDGF pathways. Docking studies suggested that myriocin can fit in the binding pocket of two target proteins (AKT1 and MAPK8).  Active ingredients of cordyceps cicadae such as myriocin may act on DN through different targets such as AKT1, MAPK8, and TP53 and other targets, which can help to develop innovative drugs for effective treatment of DN. Diabetic nephropathy (DN), also known as diabetic kidney disease (DKD), is one of the most common secondary nephrosis with high incidence rate and low cure rate. DKD develops in approximately 40% of patients who are diabetic and is the leading cause of chronic kidney disease (CKD) worldwide . It incrCordyceps cicadae, also called “Chan Hua,” belongs to the family Clavicipitaceae, Ascomycotina, and its anamorph is Isaria cicadae Miq. Furthermore, cordyceps cicadae has been used as a substitute for cordyceps sinensis . CordyceNetwork pharmacology, which is aimed at studying the complex, diverse relationships between targets, drugs, diseases, and pathways, presents a new approach for drug discovery . NetworkThis study is aimed at exploring the therapeutic effects and mechanisms of cordyceps cicadae on DN by the network pharmacology approach. The workflow is shown in http://tcmspw.com/tcmsp.php). On account of that, cordyceps cicadae contains almost all the same active components of cordyceps sinensis; the keywords are “Cordyceps sinensis” and “cordyceps cicadae” so that the eligible active components and corresponding targets are collected. Symmap database (http://www.symmap.org/) was used to search all the active components and targets with the keywords of “Cordyceps sinensis” and “cordyceps cicadae.” The compounds and corresponding targets with OB ≥ 30% were screened out, and the repetitive compounds were removed. Moreover, PubMed (https://pubmed.ncbi.nlm.nih.gov) was used to search the literature for further information. Finally, the candidate active compounds were identified.Most components are collected from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database (http://pubchem.ncbi.nlm.nih.gov), (2) TCMSP database, (3) Symmap database, and (4) PubMed database.The targets were obtained from four aspects: (1) PubChem database (http://ctdbase.org/about/) to search genes related to diabetic nephropathy.With “diabetic nephropathy” as keywords, the CTD database was used . The intersection targets of candidate compound and diabetic nephropathy were imported into the STRING database (https://string-db.org/) to obtain protein interaction information. The condition was limited to “Homo sapiens.” The confidence score with correlation degree was set to ≥0.700. The PPI data was imported into the software Cytoscape 3.6.0 to construct the network diagram of intersection target protein interaction.The intersection targets of candidate compound and diabetic nephropathy were obtained by using the omicshare website . The proteins were prepared using the Prepare Protein protocol in DS3.5, to remove all crystallographic water, add hydrogen atoms, repair broken chains, and add CHARMm force field. A sphere binding site was generated using the define site tool in DS3.5. Before docking, the small molecule myriocin was prepared using the Prepare or Filter Ligands protocol in DS3.5. The CDOCKER protocol in DS3.5 was used to perform the molecular docking. The images were created by PyMOL.Docking studies were finished by Discovery Studio 3.5 to explore the predicted binding modes of myriocin in AKT1 (PDB code: 3OCB) and MAPK8 (PDB code: 4G1W), respectively, which were downloaded from RCSB Protein Data Bank , an atypical amino acid, was isolated from the culture broth, mycelia, and sporoderm-broken spore powders of cordyceps cicadae [This study systematically studied the potential mechanism of cordyceps cicadae for the treatment of DN by the method of network pharmacology and molecular docking analysis and analyzed and constructed the “active ingredient-target-disease” network diagram of cordyceps cicadae for the treatment of DN. 36 main active ingredients of cordyceps cicadae and 85 common targets of DN, including myriocin, guanosine, and adenosine, were screened out through the database search and screening. Besides, key targets such as AKT1, MAPK8, MAPK1, TP53, IL6, and TNF were screened out. Molecular docking studies showed the binding modes of myriocin with ATK1 and MAPK8. AKT is a serine threonine kinase, a central regulator of cell growth, proliferation, survival, and metabolism. Podocyte injury is a predictive indicator of DN. It has been reported that can inhibit podocyte autophagy and aggravate its apoptosis through the AKT pathway . IL-6 isr matrix . MAPK8 ir matrix , and lipr matrix . Mitochor matrix . The res cicadae . The molIn conclusion, our study predicted target proteins related to candidate compounds of cordyceps cicadae by network pharmacology and verified by molecular docking. Since traditional Chinese medicine contains a variety of active compounds, they can act on multiple targets and signal pathways at the same time and produce a synergistic effect, which can be an effective method for the treatment of DN. Moreover, we can find particularly active compounds of traditional Chinese medicine. It is possible to develop innovative drugs for the treatment of DN diseases and to break through the difficult problems in the treatment of DN.


Fusarium lateritium. Aiming to increase knowledge about this fungal pathogen, the whole-genome sequencing of a strain isolated from symptomatic hazelnut was performed using long Nanopore reads technology in combination with the higher precision of the Illumina reads, generating a high-quality genome assembly. The following phylogenetic and comparative genomics analysis suggested that this isolate is caused by the F. tricinctum species complex rather than F. lateritium one, as initially hypothesized. Thus, this study demonstrates that different Fusarium species can infect Corylus avellana producing the same symptomatology. In addition, it sheds light onto the genetic features of the pathogen in subject, clarifying facets about its biology, epidemiology, infection mechanisms, and host spectrum, with the future objective to develop specific and efficient control strategies.In summer 2019, during a survey on the health status of a hazelnut orchard located in the Tuscia area , nuts showing symptoms, such as brown-grayish spots at the bottom of the nuts progressing upward to the apex, and necrotic patches on the bracts and, sometimes, on the petioles, were found and collected for further studies. This syndrome is associated with the nut gray necrosis (NGN), whose main causal agent is  Corylus avellana L. (hazelnut) is a shrub species belonging to the Betulaceae family. Italy is the second largest hazelnut producer in the world, with an average production of about 140,000 t/year spread among four regions , behind Turkey . In the last decades, a fruit rot causing considerable yield losses has been observed and described as a new disease. The symptomatic fruits were characterized by the presence of brown-grayish spots at the bottom of the nuts progressing upward to the apex, and necrotic patches on the bracts and, less often, on the petioles  Sacc.] as its causal agent  was carried out in order to combine an agronomic evaluation of the state of the field approach with a Supervisory Control and Data Acquisition (SCADA) system for the precision farming of orchards. Nuts showing NGN symptoms were found and collected for further laboratory analysis and molecular characterization.Fusarium genomes showed that, despite a morphological similarity with F. lateritium, this isolate is related to the F. tricinctum species complex rather than to the F. lateritium one. Thus, this study aims to provide new insights about the complexity of Fusarium species that infect tissues and fruits of hazelnut and to better understand the genetics behind the pathogenic mechanisms of this fungal strain. The new information achieved represents the basis for a better focused and effective control strategy of this disease.Aiming to increase knowledge about this fungal pathogen, the genome of one fungal strain isolated from a typical NGN diseased nut was sequenced using both long- and short-reads sequencing technologies. The resulting genome when compared with other available 2 were taken from the nut surface and placed onto potato dextrose agar (PDA) plates supplemented with 0.2 g⋅L–1 streptomycin sulfate. The Petri plates were then incubated at 25°C until the fungal colonies were grown enough to be singularly further transferred onto new PDA plates to finally obtain monosporal isolates.Symptomatic nuts (Tonda Gentile Romana cv) were collected from a mature hazelnut orchard located in the Viterbo area (VT) . Small fragments of approximately 2 mmThe Fusarium Laboratory Manual  . After 1y Manual .genus/species level, two genes were amplified using two different pairs of primers, one targeting the internal transcribed spacer (ITS)  for DNA extraction. After 5 min of centrifugation at 12,000 rpm, the supernatant was transferred into a new microtube, and the DNA was precipitated with isopropanol and 70% ethanol. To characterize the isolates at the er (ITS)  and the  1-alpha , and theFusarium sp. was grown on SNA at 25°C for 10 days (6 spore/ml. Inoculations were performed using two techniques: (i) inoculation of young to fully formed fruits with 20 μl of conidial suspension (106 conidia/ml) dropped between nut and bracts; and (ii) inoculation of the hazelnut mesocarp with 20 μl of conidial suspension (106 conidia/ml). Control fruits were inoculated with water. Inoculated and control fruits were placed in a moist chamber at room temperature for 12 days. The appearance of any symptoms was monitored daily.To confirm that the isolate in subject was responsible for the observed symptoms on fruits, the following pathogenicity tests were carried out. For inoculum preparation, the PT isolate of the  10 days . Then, c6 conidia inoculated in 100 ml of Czapek Dox Yeast broth  and 5 μl proteinase K (20 mg/ml) were added to the sample and the solution was gently mixed and incubated overnight at 56°C. After 5 min of agitated incubation with 500 μl of phenol:chloroform:isoamyl alcohol (25:24:1) and 10 min of 12,000 rpm centrifugation, the aqueous clear phase was transferred to a new tube and incubated with 500 μl of chloroform:isoamyl alcohol (24:1). After 10 min of centrifugation at 12,000 rpm, the aqueous phase was collected into a new tube and 0.6 VOL of cold isopropanol was added, followed by 30 min of centrifugation at 12,000 rpm for the DNA precipitation. Two cleaning/precipitation steps using 1 ml of 70% cold ethanol were performed by 10 min of centrifugation at 12,000 rpm. The pellet was dried, resuspended into a 100 μl of ultrapure sterile water, and treated with RNase at 37°C for 30 min. The DNA integrity was evaluated on a 1% agarose gel electrophoresis run, whereas the DNA purity was checked using Nanodrop™ spectrophotometer (Thermo Fisher Scientific). DNA sequencing was performed on a MinION Mk1b device  using a R9.4.1 Flow Cell (ONT), after library preparation using the SQK-RBK004 Rapid Barcoding Kit (ONT). An aliquot of the same DNA sample was sequenced at Eurofins Genomics  with the genome sequencer Illumina NovaSeq 6000 S2 using the paired-end sequencing.Illumina reads quality was evaluated using FastQC , NovaSeqhypocreales_db10 as ortholog lineage dataset, which consists of a set of 4,494 conserved profiles.The obtained assemblies were further polished using Nanopolish version 0.11.1 , Racon vde novo MAKER pipeline version 3.01.03  were given as gene models, and the maximum intron size was set as 2,500. Functional annotation was performed using BLASTp and SwissProt as a database.The draft genome assembled using Canu, chosen for downstream analysis, was structurally annotated following the  3.01.03 , using tF. culmorum, F. circinatum, F. oxysporum f. sp. koae 44, F. pseudograminearum CS3270, F. solani IlSc-1, F. tricinctum INRA104, F. tricinctum NRRL25481, F. tricinctum T6, F. verticilloides BRIP 53263, BRIP 53590, and all the F. avenaceum isolates, except for the already annotated Fa05001 used for the following comparative genomics analysis.The same annotation pipeline was applied to annotate the genomes of Fusarium species, together with their annotated proteins when available, were downloaded from the NCBI genome databases and used for phylogenetic and comparative genomics analysis , a phylogenetic ML tree was built on the alignment of the concatenated sequence of the DNA-directed RNA polymerase II largest (RPB1) and second largest subunit (RPB2) nucleotide sequence, as previously applied by Fusarium isolates used in this analysis are reported in For a phylogenetic analysis, the gene sequences of six housekeeping genes  were extracted from each genome and concatenated and aligned using MUSCLE . The res strains . To bettOrthologous proteins were identified using OrthoFinder, and the results were used to build a species tree, visualized using FigTree as well . The aveThe putative secreted proteins involved in pathogenesis were identified using SignalP version 5.0b with the cutoff set ≥ 0.5 Almagro . TargetPFusarium species  (The biosynthetic gene clusters (BGCs) were automatically searched and analyzed using AntiSMASH6 . The pre01343.1) .Fusarium sp. strain was isolated from symptomatic nuts collected from a hazelnut field located in the VT , which was being monitored in the framework of the PANTHEON project and named as Fusarium sp. isolate PT , with a mean read length of 2,740 bp and an N50 of 4,588. Illumina NovaSeq 6000 S2 sequencing produced 2 × ∼2.5 M reads .After low-quality reads and adapters removal done by sickle and trimmomatic, respectively, the total number of the paired-end Illumina reads resulted to be 4.8 M in total, with an average length of 151 bp and a GC content of 47%. Instead, the average length of the Nanopore reads, affected by the starting DNA fragments material, resulted in an average length of 2,740 bp, with a GC content of 47%.The reads were assembled following several pipelines and, thus, different algorithms whose results are described by QUAST statistics . SPAdes Then, the completeness of the three best quality assemblies was further evaluated by looking for the presence of 4,494 conserved ORF among the Hypocreales order (BUSCO). In fact, the polishing steps were needed to increase the quality and completeness of each of the three assemblies as well as to reduce the fragmented or missing BUSCO, leading to 99.6, 99.8, and 99.5% of completeness, respectively .JAHMRZ000000000.Based on the assembly statistics results, the draft genome assembled using Canu was chosen for downstream analysis. Its sequence was further verified by mapping the Illumina reads back on the 27 contigs used as reference to finally reach a high-quality consensus sequence  of the dentical . On the milarity .Fusarium strains, a ML tree was built on 4,319 SNPs found in the core genome alignment derived from Panseq. As shown in Fusarium sp. isolate PT within the FTSC was confirmed. The same clusterization was obtained when the orthologous proteins were identified and a species tree was built using OrthoFinder  and 79 genes encoding glycosyltransferases (Gts) . Among tSeventeen biosynthetic gene clusters belonging to T1PKSs, NRPs, and terpene synthases (Tss) were found using antiSMASH 6. Among the T1PKSs gene cluster, the fujikurin A, B, C, and D were found with 83% of similarity, bikaverin with 42 or 57% of similarity, and fusarielin H and oxyjavanicin with 50% of similarity. Besides gibepyrone A with 40% of similarity, the ACT-Toxin II shared 100% identity with the sequence deposited in the database. The NRP gene cluster is represented mainly by ilicicolin H (50% of similarity) and chrysogine (83%), whereas BGCs belonging to bassianolide, beauvericin, and fusariodione A showed a similarity below 20%. Finally, the koraiol and α-acorenol terpenes showed 100% of similarity, whereas squalestatin S1 and gibberellin were of 40% similarity.F. scirpi and F. tricinctum strain INRA104, respectively  technologies are a powerful tool to solve these misunderstandings. Furthermore, they do not only allow accurate phylogenetic analysis but also provide the basic for a thorough understanding of molecular pathogenetic mechanisms involved in the plant-pathogen iteration, like the identification of the effector genes . KnowledFusarium sp. isolate PT isolated from hazelnut in Central Italy, which initially was thought to be F. lateritium, due to the previous knowledge about the symptoms induced in the host and the morphology of the pathogen itself  or alone: (i) Miniasm-Minimap2, which use the overlap-layout-consensus (OLC); (ii) SPAdes uses the de Bruijn graph; (iii) MaSuRCA combines the de Bruijn graph with OLC, creating intermediate super-reads, further polished with POLCA; and (iv) Canu follows a MinHash Alignment Process (MHAP) based on k-mer weighting.Fusarium world  with the highest N50  without Ns. In literature, good performance of Canu have been reported in F. musae .In Oregon, Fusarium species undoubtedly have a key role in this type of disease. What remains to be assessed is the specific relevance of each of these species in the disease progression and which environmental factors influence the evolution of the disease. Investigating genomes as we did in this study, beside shedding light in the taxonomy of fungal species, particularly significant for the complex genus Fusarium, should allow to identify the genetic features of the pathogens involved in pathogenicity, thus representing milestones in understanding its evolution and eventually plan efficient control strategies to protect an important Italian crop as C. avellana.All these studies demonstrate unequivocally that several fungi can concurrently invade and damage hazelnut generating various expressions of external and internal defects. Among them, The Genome sequence can be found on the NCBI database genome under the accession number provided. The raw sequencing data are available under request.ST performed all the bioinformatics analysis, interpreted the results, prepared the figures, and wrote the manuscript in consultation with MD, LF, and AM. AG conceived and designed the study, collected the samples, and participated in the analysis. MD and CD performed the lab experiments. LF and MR provided the critical feedback and helped shape the research. VC and AM supervised the project and approved all the analysis. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The assessment of the actual impact of discharged wastewater on the whole ecosystem and, in turn, on human health requires the execution of bioassays. In effect, based on the chemical characterization alone, the synergistic/antagonistic effect of mixtures of pollutants is hardly estimable. The aim of this work was to evaluate the applicability of a battery of bioassays and to suggest a smart procedure for results representation. Two real wastewater treatment plants were submitted to analytical campaigns. Several baseline toxicity assays were conducted, together with tests for the determination of endocrine activity, genetic toxicity and carcinogenicity of wastewater. A “traffic light” model was adopted for an easy-to-understand visualization of the results. Although the legal prescriptions of chemical parameters are fully complied with, bioassays show that a certain biological activity still residues in the treated effluents. Moreover, influent and effluent responses are not always appreciably different. Some tests employing human cells were revealed to be only partially adequate for environmental applications. An interesting and helpful development of the present approach would consist in the estimation of biological equivalents of toxicity, as shown for the estrogenic compound 17-β-estradiol. Water policies have been changing progressively during the recent decades, keeping up with the awareness of the need to save and preserve this resource.The protection of water resources  goes far beyond mere compliance with the existing legal limits and guidelines: a radical change of perspective has been increasingly needed, leading to water resources being considered as part of complex ecosystems, where abiotic factors and biotic components coexist. Consequently, the entire supply chain, from the catchment of water for human consumption to its discharge into the environment, is experiencing progressive improvements, with the final aim to decrease the anthropogenic impact on waterbodies.From this point of view, a careful and deep evaluation of potential effects of the Waste Water Treatment Plant (WWTP) effluents discharged into surface water, on both ecosystem and human health is of primary importance. WWTPs, in effect, may represent hotspots for those trace pollutants collected by sewers: the upgrading and development of efficient treatment technologies and the implementation of adequate process schemes represent the first response to this issue. The choice of the optimal solutions, however, must be based on the knowledge of the real impact of a plant ,2,3.It is widely recognized that a wastewater characterization focused only on chemical analyses provides a partial representation of the effects of a WWTP effluent. Several studies, indeed, have shown that only an integrated monitoring based both on chemical analyses and biological assays can yield to a more realistic evaluation of sewage degradation and detoxification performance of a WWTP ,5,6,7. IThe aforementioned statements, which are still valid for mixtures, are even more important in case of wastewater treatment processes, where physical, chemical and biological transformations and decompositions lead to the production of new substances.The results of an integrated monitoring, based on chemical analyses and toxicological tests, could be successfully integrated in the environmental footprint evaluation based on a Life Cycle Assessment (LCA) approach, which represents a useful tool for evaluating and benchmarking the actual impact of products and organizations ,9.Notwithstanding the general scientific consensus about the invaluable role of bioassays in environmental toxicity assessment, and the host of projects/studies/working groups  bioassays, which are shown to be a powerful tool for understanding the real interactions of matrices discharged into the environment. Even more importantly, an attempt for overcoming ambiguity and misinterpretation of the results is presented. Common toxicological tools, such as tests performed on crustaceans, algae and luminescent bacteria, have been designed for evaluating effluent quality for legal purposes. The assessment of the real impact of wastewater discharge, however, should include a wide range of different biological target . TherefoThe municipal WWTPs chosen as case studies are located in the North of Italy. WWTP A  treats domestic wastewater with a remarkable contribution of industrial discharge from the agro-food sector. WWTP B  treats important amounts of winery effluents, which, during the grape harvest time (September and October), increase the pollutant influent loads, respect to the “routine” period. Both plants adopt the conventional activated sludge process. A detailed description is reported in the following paragraphs , pre-denitrification (7140 m3), oxidation/nitrification , secondary settling . Excess sludge undergoes dynamic thickening, anaerobic digestion and mechanical dewatering. The typical influent flowrate is equal to 74,900 m3/d . Unlike the flowrate, the wastewater composition varies during winter and summer periods, due to the contribution of the seasonal industrial activities (agro-food manufacturing sector). Typical concentrations of the main pollutants are the following, in the summer and winter periods, respectively. Standard deviations are reported between brackets. COD: 300 (42) and 540 (85) mg/L; BOD5: 170 (30) and 300 (63) mg/L; total nitrogen: 45 (8) and 70 (12) mg/L; total phosphorus: 5.0 (0.8) and 7.2 (1.4) mg/L. Effluent concentrations are rather stable all along the year as show by the following data. COD: 22.7 (8.0) mg/L; BOD5: 5.8 (2.5) mg/L; total suspended solids: 8.5 (5.8) mg/L; total nitrogen: 14.5 (5.6) mg/L; total phosphorus: 1.5 (0.9) mg/L. The sludge retention time in the aerated reactor is equal to 8 ± 2 d; wastewater temperature ranges between 15 and 26 °C .WWTP A consists of: pre-treatments , primary settling , preliminary treatments , pre-denitrification (2160 m3), oxidation-nitrification (7120 m3), final sedimentation (4564 m3), disk filtration, UV disinfection. The sludge treatment line consists of dynamic thickening and mechanical dewatering. The main influent characteristics in the routine and grape harvest periods, respectively, are reported in the following (standard deviations between brackets). Flowrate: 20,300 (2500) and 26,170 (3100) m3/d; COD: 290 (32) and 430 (75) mg/L; BOD5: 130 (18) and 220 (40) mg/L; total nitrogen: 17 (2.0) and 18 (1.9) mg/L; total phosphorus: 2.5 (0.3) and 3.3 (0.4) mg/L. As for the effluent, the typical characteristics of the two periods are the following. COD: 17.3 (5.2) and 19.9 (6.3) mg/L; BOD5: 6.9 (3.2) and 8.0 (4.1) mg/L; total suspended solids: 6.9 (5.3) and 7.3 (6.0) mg/L; total nitrogen: 5.8 (3.0) and 5.0 (3.2) mg/L; total phosphorus: 0.5 (0.3) and 0.4 (0.3) mg/L. The sludge retention time is kept between 15 and 25 days in aerated reactors. Wastewater temperature ranges between 10 and 25 °C.The WWTP B treatment train includes the following units: equalization . Moreover, different levels of biological organization were targeted  as well as different biological complexity . Finally, the bioassays can detect both baseline toxicity and particular modes of action, adopted in order to correlate key events with biological answers. The modes of action (MOA) explored in this toxicological study, as well as the specific tests and the measured phenomena are the following:The experimental methodologies are described at length elsewhere .Chemical analyses were addressed to the determination of conventional parameters  and the quantification of inorganic and organic pollutants, to better characterize the quality of the streams. Selected conventional parameters are: total suspended solids, biochemical oxygen demand (BOD), chemical oxygen demand (COD), total nitrogen, total phosphorus. These pollutants were analyzed daily. Boron, vanadium, chromium, manganese, iron, nickel, copper, arsenic, selenium, cadmium, antimony, aluminum, mercury, lead were analyzed weekly. Herbicides, insecticides and degradation by-products, perfluorinated alkyl substances, polynuclear aromatic hydrocarbons were also determined once a week. Details of analytical methods are reported in The integrated and overall examination of the results requires data harmonization, in order to have a comprehensive view of possible toxic effects exhibited by the tested samples. Thus, a “traffic light” visualization was proposed. A color  was attributed as a function of biological response intensity: the criteria underpinning the threshold settings are widely explained in An important information to take into account for the correct interpretation of results is the correlation between the biological response and the degree of dilution (for those test conducted on raw samples) or concentration (when extracts are used) of the samples. In case of dilution of the raw wastewater the calculation is quite simple. On the contrary, when sample pre-treatment is required, both dilution and enrichment processes (the latter consisting in a solid phase extraction—SPE) must be taken into account.REF) [SPEEF) and the actual sample dilution in the bioassay (dilution factor: bioassayDF) (Equation (1)).In this case, the range of sample concentrations tested in the different bioassays was expressed in unit of Relative Enrichment Factor (REF) ,37, whicSPEEF was calculated using Equation (2) as the ratio between the volume of processed water (waterV) to the volume of resulting extract in solvent (extractV).The The dilution factor of each tested dose was calculated using Equation (3).REF equal to 1 (REF 1 sample) means that organic analytes concentration in the bioassay is equivalent to that of the unprocessed water . On the contrary, a value higher, or lower, than 1 expresses, respectively, a sample concentration or dilution.Thereby, a value of SPEEF and the final volumes considered for each bioassay are reported in The values of The main influent and effluent characteristics are summarized in 50 values reported in literature for D. magna. Only for copper and mercury the measured concentrations are relatively close to the respective EC50.During the monitoring campaign, all the measured polynuclear aromatic hydrocarbons, chlorinated insecticides and herbicides were below their detection limits, which are reported in Baseline toxicity exerted on daphnids, algae and bacteria was not decreased after the treatment: detailed results of these bioassays are reported in the Allium cepa test showed toxicity in influent undiluted sample: the roots elongation was negatively influenced  for influent is twenty times higher than for the effluent, demonstrating the positive effect of the plant treatments on cell viability and thus the noteworthy reduction of toxic potential of this wastewater. The REF 1 effect was extrapolated by dose-response curves: no toxicity was estimated  was used. Using the standard curve elaborated with 17β-estradiol (E2) see , the endSalmonella, in both strains, with and without exogenous metabolic activation , are presented in The results of comet assay are reported in Allium cepa roots at all tested dilutions  was used. Beside the negative control, cells were treated with TPA (positive control), a well-known inhibitor of communication mediated by gap junction (GJ) and a reference tumor promoter. As shown in A. fischeri and R. subcapitata) show an apparent worsening of the effluent, with respect to the untreated wastewater. Indeed, the quantified bacteria luminescence inhibition passed from 48% (influent) to 56% (effluent), which is a rather small or even not significant difference, considering the uncertainty of the bioassay. Likewise, and even less significantly, the algal growth inhibition increased from zero to only 11% (10% being the minimum threshold causing the yellow color attribution). On the contrary, the A. cepa assay shows an appreciable quality improvement, corresponding to a two-classes jump (from red to green) after biological treatment. It is interesting to compare these results with legal limits established for defining an effluent as acceptable. In particular, the Italian regulation states that less than 50% immobilization/inhibition is to be caused . Under this perspective, only the bacteria reveal particularly sensitive, after exposure to both influent and effluent, whose effects remain almost unchanged after the treatment. On the contrary, crustaceans and green algae are not significantly affected, thus describing good quality samples.First of all, the importance of using different tests, within the same mode of toxic action, has to be underlined. In effect, responses to different organisms should be compared together, to obtain reliable results. As for baseline toxicity, for example, two tests . Indeed, the estimated equivalent E2 concentration of the REF 1 sample resulted much higher than the reference values for groundwater recharge and drinking water. And this is very interesting considering that measured organic pollutants concentrations were extremely low.Perna viridis and Corbicula fluminea). Several species, in addition, could be employed in a passive monitoring, placed in situ, for instance downstream the effluent discharge point. The exploitation of Comet test as ecotoxicological tool must overshoot the interpretative criteria of the biomedical research and human toxicology; proper models, chosen based on ecological roles and life cycle (stages), would provide more accurate information on freshwater ecosystem impacts.Regarding genetic toxicity, all performed tests show no difference between the influent and effluent samples. In particular, green color has been always attributed, apart from the case of comet assay. Interestingly, the damage occurred to DNA of human leukocytes might be reassessed based on the inherent role played by this assay for the environmental impact assessment. Indeed, the comet test does not allow detection of the DNA fragments, which originate from apoptosis and necrosis: therefore, cytotoxicity may possibly lead to false positive and negative results . Surprisingly, influent and effluent samples did not show appreciably different behaviors.The main influent and effluent characteristics are summarized in 50 towards D. magna , as well as the ECagna see . As in tR. subcapitata test shows a different result for the first campaign samples; notably, this is the only pejorative result out of six, thus not modifying the overall judgement. It can be observed that the influent wastewater toxicity measured during the second campaign is higher. One reason could be the relevant concentration of surfactants, whose toxic action is known [All the conventional baseline toxicity tests prescribed by the law demonstrate the compliance of the effluent in both campaigns (detailed data in is known .Allium cepa exhibit no toxicity in both samples of the first campaign (green light). Conversely, during the spring campaign, the influent sample showed a slight toxicity (yellow), which disappeared after biological treatment (green) (see Influent and effluent tested on een) see . The sub50 for the influent is about ten times higher than for the effluent, demonstrating the positive effect of the polishing treatment. Indeed, the IC50 is achieved only at very high concentration factors, while the chromatic evaluation refers to the REF 1 condition.Cytotoxicity (MTT test carried out on IAR203 hepatocytes with REF 1 sample) reveals absent in both periods, thus the green color was attributed. Nevertheless, as shown in As mentioned above, a non-cytotoxic dose of extracted wastewater (REF 20) was used for assessing the estrogenicity. Using the standard curve , revealing the presence of directly active mutagens causing frameshift mutation, also indicating the detoxifying action of the S9 . For thiSalmonella typhimurium strains treated with and without enzymatic activation have a similar behavior in terms of genetic damages. Anyway, influent taken during grape harvest time exhibits a slightly weaker toxicity with respect to other samples.All the samples collected at the WWTP B show damages to the DNA of exposed cells leading to red color attribution see . EffluenAllium cepa genotoxicity tests (4.4% vs. 2.5% negative control and 3.3% effluent). No other sample was able to induce DNA damage (green light), as either chromosomal aberrations or micronuclei , using As for the other tests, the in vitro transformation assay was performed after the preliminary cytotoxicity assessment, as detailed in .p < 0.05) is clear in samples at REF 31.25. The significance threshold (p < 0.05) suggests a yellow level of attention (see p < 0.01.The mean number of transformed foci (Type II and III) generated after the exposure to the samples is shown in tion see . WhereasAs shown in D. magna test, A. fischeri test, A. cepa and MCF-7 cells estrogenicity decrease, at least for one campaign, after the wastewater treatment. On the contrary, other assays, such as R. subcapitata test, Ames test, CTA and tumor promotion test show, in some cases, a more toxic effect of the effluent sample. Interestingly, the influent wastewater of the routine period induces a greater baseline toxicity, partially ascribed to the high concentration of surfactants. It has to be underlined, however, that the results marked with red Color refer to experiments conducted on heavily concentrated samples. Indeed, as for the MCF-7 test, the estimated equivalent E2 concentration of the raw sample is very low. Moreover, regarding the Comet test, the statements reported above on the adequacy and suitability of this biological model  is now under debate.The translation of all the results into a chromatic code is displayed in To sum up, the quality of the effluent can be considered as acceptable, in terms of legal prescriptions and based on the results of the majority of the proposed tests. As observed for WWTP A, only the response of human cells leads to apparent criticalities, possibly due to the partial adequacy of this biological model for environmental applications. A substantial improvement of effluent quality is obtained after the treatment.The findings obtained in this study show the opportunity to perform a series of diverse assays, given that several specific endpoints can be targeted, and different biological models chosen, thus enabling to get a possible confirmation of the results. Furthermore, some tests, as in the case of estrogenicity assessment, describe the overall behavior of mixtures of substances, which are considered with increasing attention by the policy makers and included in a growing number of legislative documents.The power of a multitiered approach suggested by several researchers has been confirmed, although further monitoring campaigns on different wastewater treatment plants are required, in order to better tune the sensitivity of the experiments and to select new ones (either replacing or adding those proposed here), with particular focus on the need to diminish the masking effect exerted by toxicity towards definite endpoints. Additionally, some tests conducted on human cells  revealed highly sensitive.Specifically, the suggested traffic light approach might help in getting a comprehensive overview. Interestingly, bioassays revealed that, in some cases, the effluent maintains a certain biological activity.The authors believe that the conventional toxicity assays should be profitably integrated with additional and complementary ones, to properly characterize the possible effects of the discharged wastewater on the freshwater biota. The estimation of biological equivalents of toxicity, obtained by testing reference substances, could provide further quantitative information, as shown in the case of the estrogenic compound E2.The outcomes of the present research are very promising, though not exhaustive, and they represent a solid starting point for further studies, which are already ongoing.


More patients with moderate-to-severe HIE had confirmed HIs  than those with mild HIE. Clinical staging, and blood lactate and glucose levels could be predictive factors for HIs among patients with HIE. The patients who exhibited HIs had significantly higher lactate  and serum glucose  levels than those without HIs. A higher prevalence of HIs was noted in the patients with stage III HIE than those with stage II HIE . The degree of HI correlated with brain anomalies and neurodevelopmental outcomes at 1 year of age. Clinical staging, and blood lactate and glucose levels could be predictive factors for HIs among patients with HIE.Identifying biomarkers for hearing impairments (HIs) in patients with neonatal hypoxic–ischemic encephalopathy (HIE), to initialize early hearing habilitation, is crucial. Seventy-eight neonates with HIE were divided into the following two groups: those with HIs and those without HIs. We compared those patients with 11,837 newborns without HIE, and analyzed the risk factors of HIs among neonatal HIE. Of the 78 patients, 11 were confirmed to have an HI, which is a substantially higher percentage than in the 11,837 newborns without HIE (14.1% vs. 0.87%;  Hearing impairments (HIs) in newborns can cause a serious obstacle to their development in language, cognition, and education ,2,3, andBirth asphyxia is a physiological disorder that can develop in newborns as a result of a prolonged or profound disparity between oxygen demand and oxygen delivery . ModeratInducing hypothermia among those patients with neonatal HIE is effective, leads to few adverse effects among newborns ,7,8, andThe risk factors of HIE with hearing loss are a history of receiving mechanical ventilation, use of ototoxic medications, admission to the neonatal intensive care unit for more than 7 days, and a low Apgar score . HoweverThe early diagnosis of hearing problems and the rapid initialization of a hearing habilitation program are vital for a favorable long-term prognosis in neonatal neurodevelopment, particularly in language and cognition. As identifying the risk factors for HIs is also valuable for managing neonatal HIE, to avoid further damage, we investigated the risk factors of hearing damage in patients with neonatal HIE. We suggest early intervention for those patients with an HI.We retrospectively reviewed the records of patients with neonatal HIE at Chung Shan Medical University Hospital, located in central Taiwan, by identifying those with a clinical history of fetal distress, metabolic acidosis, or needing positive-pressure ventilation immediately after birth. HIE was classified by clinical Sarnat staging into stages I (mild), II (moderate), and III (severe) ,7,17 by From 2012 to 2020, we performed global newborn hearing screening for a total of 11,837 newborns. Newborn hearing screening was performed by aABR after birth for all newborns. For patients who failed the aABR twice, ABR, otoacoustic emissions (OAE), and steady-state evoked potentials (SSEP) were performed ,19.The neurodevelopmental outcomes were evaluated at 1 year of age. The Bayley Scales of Infant and Toddler Development, Third Edition (Bayley-III), were adopted to evaluate patients at the age of >1 year. The Bayley-III scores were interpreted as follows: normal, ≥85; mild, 70 ≤ and <85; moderate, ≤55 and <70; severe, <55 ,21. The t-test to compare the means of two independent groups, or a chi-square test between categorical variables. The Fisher exact test was used when sample sizes were small. The odds ratio (OR), the association between a variable and an outcome, was calculated by dividing the odds of the first group by the odds of the second group. If the sample distribution was nonparametric, a Mann–Whitney U test was performed. Significance was set at p < 0.05. All statistical tests were conducted using SPSS .Significant differences between the groups were evaluated using an independent Patient charts were retrospectively reviewed in all cases. Ethical approval of the study was provided by Chung Shan Medical University Hospital’s Internal Review Board (IRB #: CS2-14003), and the study was performed in accordance with relevant guidelines.p < 0.001) than that of all the newborns tested. The presence of HIs in the 11 patients who failed the hearing screening was further confirmed by ABR, OAE, and SSEP, and 17 individual ears exhibited hearing loss, of which 8 ears had mild (>35 and  ≤45 dB nHL), 0 ears had moderate (>45 and  ≤65 dB nHL), 1 ear had severe (>65 and  ≤90 dB nHL), and 8 ears had profound (>90 dB nHL) hearing loss. The failure rate of neonatal hearing screening in newborns, from a total of 11,837 newborns in our hospital, was 103 (0.87%). Of the 78 patients with neonatal HIE, 11 did not pass the hearing screening. Therefore, the failure rate among patients with neonatal HIE was significantly higher , 30 were in stage II (moderate), and 16 were in stage III (severe). Of the stage 1 patients with neonatal HIE, 1 (3.1%), with mild HIE, had an HI in 1 ear, and 10 (21.7%) out of 46 patients with moderate or severe HIE, who were receiving hypothermia therapy, had HIs. p = 0.020; OR, 8.61; 75% CI 1.04 to 71.10). The demographic data of patients with neonatal HIE, with and without a hearing impairment, are presented in p = 0.032; p = 0.036; A higher rate of hearing loss among the patients with moderate-to-severe neonatal HIE was observed  = 5.39, n = 46) = 6.9862, p = 0.008) than those with HIs in clinical stage II  (7 of 16, 43.8%; χ2 (1, Among 11 patients with neonatal HIE and HIs, 9 had abnormal brain MRI results and 2 had unremarkable brain MRIs . The MRIThe follow-up of patients with HIs at 1 year of age revealed the following neurodevelopmental outcomes: one was unremarkable, two had mild delays, five had moderate delays, and three had severe delays. One patient with mild HIE was intubated because of lung parenchymal disease, and exhibited nystagmus and motor delays. The degree of the patients’ HIs corresponded to their degree of neurodevelopmental delay at 1 year of age. In our study, the clinical staging, and blood lactate and glucose levels measured within 6 h of birth were significantly related to the occurrence of HIs in patients with neonatal HIE. Selective neuron neurosis is a probable cause of HI because HIE may cause nuclei injury in the highly susceptible brainstem, as was observed in the brain MRIs of patients with HIE and profound HI. In our previous study , WBCs, aWe noted that the HIs in patients with neonatal HIE were often combined with other neurological irregularities, including feeding problems, facial diplegia, severe cerebral palsy, or midbrain or brainstem lesions. Brain MRIs highlighted the cranial nuclei and midbrain lesions in patients with selective neuronal necrosis as their HIE progressed. In the study, the neonatal hearing defect in the hearing screenings of all the newborns was approximately 8–9/1000, and was higher than the approximately 3–4/1000 infants ,24. ThisFew studies have reported on biomarkers correlated with HI in neonatal HIE ,26,27. IOur findings indicate that the cause of HI in neonatal HIE may be the susceptibility of the patients to eighth nucleus injury during hypoxic change. This is reflected in the profound hearing loss, and midbrain and brainstem lesions. The degree of HI is also correlated with the degree of brain injury. The early identification of HI in patients with neonatal HIE is critical to their long-term neurodevelopmental outcomes. Early MRI is strongly correlated with later neurodevelopmental outcomes, which, particularly in the thalamus and basal ganglion, was often associated with HI in our study. Isolated cyst encephalomalacia that does not involve the midbrain and brainstem causes less HI. One hypothesis is that HI is related to hypothermia therapy; however, we found that HI was associated with the degree of brain injury. Hypothermia therapy might diminish HI among patients with HIE, despite no literature having reported this finding. Early hypothermia therapy for patients with moderate-to-severe HIE is critical to the brain, and to saving hearing functions.This study has some limitations. We presented a limited number of cases of HIE and HI in the analysis of the risk factors; hence, our findings might be biased. The data were compared with those of more than 10,000 newborns without HIE, who had a background of congenital hearing loss. Therefore, studies including a larger number of patients with neonatal HIE and HI are warranted. Clinical staging, and early blood lactate and blood glucose levels, within 6 h after birth, can be predicting factors for HIs among patients with neonatal HIE. Among patients with clinical moderate-to-severe HIE, only clinical staging was a predicting factor. The severity of the HI reflected the degree of injury to the vestibulocochlear nerve nuclei in the brain. The early identification of HI in patients with neonatal HIE is critical for developing timely interventions for children’s hearing habilitation.


Previous genome-wide studies have identified an association between the rs2106261 single-nucleotide polymorphism (SNP) in the zinc finger homeobox 3 (ZFHX3) gene and an increased risk of atrial fibrillation (AF). However, this association remains controversial, since conflicting results have been reported in previous studies. We aimed to investigate the association between the ZFHX3 rs2106261 polymorphism and susceptibility to AF.A comprehensive literature search, of articles written in either English or Chinese, was conducted on various databases, including PubMed, Embase, Web of Science, the Cochrane library, Wan Fang, and CNKI, for studies performed up to August 1, 2020. Data were abstracted and pooled using Stata 14.0 software. A meta-analysis was performed on all selected studies based on ZFHX3 rs2106261 polymorphism genotypes.Nine studies, including 10,107 cases and 58,663 controls, were analyzed in the meta-analysis. In the overall population, a significant association was found between AF and the T-allelic ZFHX 3 rs2106261 SNP . In subgroup analysis, a significant association between the T-allele of rs7193343 and risk of AF in Caucasian  and Asian subgroups  was observed. However, no statistically significant association was found in African populations .The genetic variant rs2106261 SNP is associated with susceptibility to AF in Caucasian and Asian individuals, with Asian samples showing a stronger association. However, based on the current evidence, no association was found in African samples. Future studies, with larger sample sizes and multiple ethnicities, are still necessary. While the underlying mechanism responsible for these associations remains to be elucidated, it is likely that risk haplotypes affect the expression of neighboring genes.Several factors associated with increased susceptibility to AF have been identified. These include age, male gender, obesity, valvular heart disease, hypertension, myocardial infarction, and family history. The association between allele T at rs2106261 and AF has been replicated in several populations. However, conflicting results have been reported, showing that the presence of the T risk allele may not be a specific risk factor for AF. To address these conflicting results, the current meta-analysis was conducted to further evaluate the associations between the rs2106261 SNP and AF risk.Previous GWAS have shown that the single-nucleotide polymorphism (SNP) rs2106261 in the zinc finger homeobox 3 (ZFHX3) gene at chromosome 16q22 is strongly associated with susceptibility to AF.2https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=106938).The present review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. The protocol for our study was registered in the International Prospective Register of Systematic Reviews, with the registration number CRD42018106938  and 95% confidence intervals (CIs).Unpublished studies, review articles, abstracts-only articles, case reports, editorials, and studies that did not contain adequate outcomes of interest were excluded from the current analysis.Two investigators (W.Y. and Z.N.) independently screened each citation for inclusion. Two reviewers (W.Y. and Z.N.) independently reviewed the full-text of articles to determine eligibility. Discrepancies were resolved by discussion with the entire research team. The following data were extracted from the included studies: the first author, publication year, country where work was conducted, number of patients/control individuals, average age/gender distribution of patients/control individuals, source of controls, genotyping method, genotype distribution, and data necessary to conduct meta-analysis. Attempts to contact the original authors for detailed information were made if the data were incomplete or missing in the publication. Quality of prospective or case-control studies was evaluated according to the nine-point Newcastle-Ottawa Scale, and quality of cross-sectional studies was evaluated according to the Agency for Healthcare Research and Quality standards.2.3I2 index were used to calculate heterogeneity across all included studies. If P < .1 or I2 > 50%, a random-effects model was used to estimate the pooled OR (DerSimonian and Laird methods), due to the presence of significant heterogeneity; otherwise, a fixed-effects model was used (the Mantel-Haenszel method).Statistical analysis was performed with Stata 14 . Strength of the association between the rs2106261 ZFHX3 polymorphism and susceptibility to AF was assessed by combining OR and CIs. Cochran's Q statistic and the P < .05.Sensitivity analysis was performed by calculating ORs repeatedly with omission of each study to check the consistency of the overall estimated effect. Publication bias was conducted both visually (via funnel plot) and statistically (via Begg and Egger bias tests). All statistical testing was two-tailed with a statistical significance set at 33.1,10–17 The results of the search and selection process are shown in Figure The electronic search yielded 151 potentially relevant abstracts. After removing duplicates and ineligible abstracts, we reviewed the full text of 23 reports. Of these reports, 14 were excluded for not meeting inclusion criteria. Consequently, nine studies were finally selected in this meta-analysis.3.2 and another article included 2 comparisons. The sample sizes ranged from 743 to 11,639. The ethnicities of the included comparisons were Caucasian (n = 7), Asian (n = 5), and African (n = 3). Genotypic distributions in controls from most selected studies conformed to Hardy Weinberg Equilibrium. Quality of included studies was considered good according to Newcastle-Ottawa Scale and Agency for Healthcare Research and Quality standards. Results are shown as supplementary data .Table 3.3P < .001; I2 = 78.7%, P for heterogeneity (Phet) < .001). These results indicated that there was a statistically significant association between the rs2106261 SNP and AF  and Caucasian  samples. However, in African samples, no significant association was observed , and no publication bias was observed in the Egger test , which is a transcription factor with multiple homeodomains and zinc finger motifs. ATBF1 has been reported to inhibit cell proliferation, thus functioning as a tumor suppressor in several cancers. ZFHX3 is widely expressed in many tissues including the heart. In a rapid pace-induced porcine AF model, where significant structural and inflammatory changes were noted in the atria, the expression of signal transducer and activator of transcription 3 (STAT3) was up-regulated, and nuclear translocation and binding to STAT3 cis-elements of the target were increased, suggesting that STAT3 plays a critical role in the pathogenesis of AF. ZFHX3 interacts with the terminal end of the protein inhibitor of activated STAT 3, which is a specific inhibitor through binding to activated tyrosine-phosphorylated STAT3 dimers, subsequently suppressing STAT3-mediated signaling pathway.,20 Additionally, knockdown of ZFHX3 has been shown to enhance the activation of STAT3, increasing the expression of sarcoplasmic/endoplasmic reticulum calcium ATPase 2a, ryanodine receptors, and several potassium channels, leading to dysregulation of calcium homeostasis and an increase in atrial arrhythmogenesis. Overexpression of ZFHX3 reversed the above effects. Taken together, the above results indicate that inhibition of ZFHX3 might contribute to AF. Moreover, ZFHX3 has been observed to regulate protein sumoylation in different biological processes, through which it may play an additional role in the pathophysiology of AF by regulating certain related proteins. Finally, ATBF1 could interact with Runt domain transcription factor 3, with the complex translocated to the nucleus upon activation of the transforming growth factor-beta signaling pathway, thus transactivating pro-fibrotic genes, which can contribute to atrial remodeling and atrial fibrosis.,24 However, the role of ZFHX3 in the pathophysiology of AF has not been fully elucidated, and further fundamental studies are still warranted.The ZFHX3 gene is located on chromosome 16q4.2 Soon afterward, a study by Li et al showed that the minor allele of rs2106261 was related to AF susceptibility in a Chinese Han population, expanding the association between ZFHX3 and AF in individuals of non-European ancestry. This result remained significant when only isolated AF was included in the analysis, suggesting that ZFHX3 gene might play an important role in the specific pathophysiology of AF. Later, the relationship between rs2106261 and AF in Korean and Japanese populations was identified and replicated, respectively,,14 therefore confirming that the risk allele of rs2106261 was associated with AF in the entire East-Asian population. Overall, rs2106261 is associated with susceptibility of AF in large populations, including individuals of East-Asian and European descent.The rs2106261 SNP of the ZFHX3 gene was first identified as a locus for AF by GWAS. This risk variant was first discovered to be significantly associated with AF in 5 community-based cohorts in the United States and Iceland, with results replicated in a Germany cohort.4.3 Taken together, these results suggest that ethnicity is of great importance in polymorphism distribution of ZFHX3 gene. Moreover, the association of rs2106261 risk allele and increased risk of AF is significant in Asian and European populations, but not in African populations. A previous meta-analysis observed that rs7193343 was associated with AF in Caucasian populations, but not in an Asian population. Potential reasons for these discrepancies are as follows:Ethnicity affects not only minor allele frequency (MAF) but also the association of the minor allele and susceptibility to AF. In our study, it was found that the distribution of the rs2106261 SNP genotype in ZFHX3 gene differs among populations. The MAF of rs2106261 ranges from 17% to 22% in Caucasian populations and has been found to be approximately 25% in African populations. However, the MAF in Asian populations was found to be above 30%. Similarly, the MAF of rs7193343, another SNP of the ZFHX3 gene, is observed more frequently in individuals of Chinese descent than of individuals in a Caucasian population.1.;inconsistency of genetic background between ethnicities2.; anddifferent lifestyle and/or diet practiced by various ethnicities3.different gene-environment interactions under different regional environmental conditions.Further genetic studies are necessary to better understand the precise underlying mechanisms responsible for these differences.4.4 was the first study that tested the effect of 2 SNPs (rs2200733 and rs10033464) on chromosome 4q25, which had been previously associated with AF. The study examined short-term and long-term outcomes after AF catheter ablation and found that the presence of either SNP increased the risk for both early and late recurrence of AF. A meta-analysis validated the fact that the rs2200733 polymorphism increases the risk of AF recurrence after catheter ablation. As for the rs2106261 polymorphism, it has been reported that it was not associated with AF recurrence after radiofrequency ablation,32 or after cryoballoon ablation. In these studies, both paroxysmal and persistent AF patients were enrolled, and strategies for ablation were multifarious. However, Tomomori et al revealed that the minor allele of rs2106261 is associated with long-term recurrence in paroxysmal AF in patients undergoing pulmonary vein isolation. In addition, Parker found that the minor allele of rs2106261 was a marker of a good response to radiofrequency ablation in long-standing AF. One possible explanation is that ZFHX3 positively regulates expression of paired-like homeodomain 2c, which is necessary to differentiate pulmonary mesenchyme into myocardium, expanding by proliferation to form the pulmonary myocardial sleeve. Therefore, the minor allele of the rs2106261 ZFHX3 polymorphism might modulate the expression of paired-like homeodomain 2c, promoting the proliferation of the myocardial sleeve around the branches of the pulmonary vein, increasing automaticity, which then serves as an AF trigger.,35 Under this circumstance, AF is related to the pulmonary vein and thus could be cured by pulmonary vein isolation. Further studies are still warranted to determine the association of rs2106261 polymorphism and outcome of AF ablation and its potential underlying mechanism.Catheter ablation, which is widely used in clinical practice, is recommended as the treatment of drug-resistant, symptomatic AF. In recent years, studies have shown that several SNPs associated with AF could predict AF recurrence after AF ablation. A study by Husser et al5The current study has a number of limitations. First, in our analysis, significant heterogeneity was observed in the overall population. After subgroup analysis was performed, no heterogeneity was found in the African population, while a trend of decreasing heterogeneity was seen in the European population, and heterogeneity remained significant in the Asian population. Nonetheless, we used sensitivity analysis methods in the random-effects model and found no difference in risk ratio or its 95% confidence interval. The heterogeneity may have been caused by1.variation in participant characteristics  existed between studies;2.type, duration, and etiology of AF were different among studies;3.underlying haplotype structure was different between study populations; or4.study-specific precision was low in some of the studies included.Second, all of the studies were published in English. Studies in other languages or unpublished studies were not included in this meta-analysis, which may have resulted in selection and/or publication bias. Third, the genotyping data of rs7193343 SNPs were insufficient, so we were unable to conduct meta-analysis based on the limited data available from the studies analyzed. Fourth, in the African population subgroup, the total sample size of AF patients was much less than that observed in the other 2 populations. More studies with larger sample sizes are needed to confirm the current conclusions. Fifth, other factors have also been associated with AF risk . The results would be more precise if individual data were adjusted controlling for these variables.6The genetic variant rs2106261 SNP is associated with susceptibility to AF in Caucasian and Asian populations, although this association is more significant in Asian populations. However, the current data show no association in African populations. Further studies with larger sample sizes and multiple ethnicities are still warranted in the future.www.letpub.com) for its linguistic assistance during the preparation of this manuscript.We thank LetPub (Conceptualization: Yue Wei, Lingjie Wang.Data curation: Yue Wei, Lingjie Wang, Ning Zhang.Formal analysis: Yue Wei, Lingjie Wang, Ning Zhang.Funding acquisition: Yangyang Bao, Ning Zhang.Investigation: Yue Wei, Lingjie Wang.Methodology: Yue Wei, Lingjie Wang.Project administration: Yue Wei, Lingjie Wang, Yangyang Bao.Resources: Yue Wei, Changjian Lin, Yun Xie, Yangyang Bao, Qingzhi Luo.Software: Yue Wei, Changjian Lin, Yun Xie, Yangyang Bao, Qingzhi Luo.Supervision: Yun Xie, Yangyang Bao, Qingzhi Luo, Ning Zhang.Validation: Changjian Lin, Yun Xie, Yangyang Bao, Qingzhi Luo.Visualization: Changjian Lin, Yun Xie, Yangyang Bao, Qingzhi Luo.Writing – original draft: Yue Wei, Lingjie Wang.Writing – review & editing: Changjian Lin, Yun Xie, Yangyang Bao, Ning Zhang.