Black-skinned men with higher SES, living alone, and with higher PS and INT levels presented higher prevalence values of CMDs. Conclusions: High levels of PS and INT were the factors that presented the strongest associations with the occurrence of CMDs among the men. It is necessary to implement actions to reduce the stress-generating sources as well as to promote an increase in resilience and the development of intrinsic reinforcements to deal with uncertain threats.Objective: To analyze the relationships between sociodemographic variables, intolerance to uncertainty (INT), social support, and psychological distress  and perceived stress (PS)) in Brazilian men during the COVID-19 pandemic. Methods: A cross-sectional study with national coverage, of the web survey type, and conducted with 1006 Brazilian men during the period of social circulation restriction imposed by the health authorities in Brazil for suppression of the coronavirus and control of the pandemic. Structural equation modeling analysis was performed. Results: Statistically significant direct effects of race/skin color (λ = 0.268;  The health crisis caused by COVID-19 exerted a negative impact on the mental health of the world’s population. The experience of unknown situations and the fear of contamination imposed by the burden of the disease and its repercussions produced new daily stressors and exacerbated previous ones already structurally present in societies ,3,4. TogIt is estimated that nearly one billion people are affected by some mental disorder due to the pandemic, with an emphasis on symptoms of anxiety, anguish and depression, and disorders resulting from alcohol use and substance abuse in addition to the increase in suicide rates ,3,4,5,6.The rapid spread of COVID-19, associated with the need for social distancing, forced quarantine, and blockades across the world, has placed imposed high stress levels on people ,15, and Consequently, the COVID-19 pandemic resulted in a psychologically chaotic and dismal setting. The radical changes in the individuals’ daily routines and the general context of fear and insecurity converge to unfamiliar situations and significant uncertainties that, due to their persistence over time, also provoke reactions of intolerance to this uncertainty ,7. IntolThus, intolerance to uncertainty is considered as one of the main underlying components of anxiety disorders , obsessiSocial support is a basic human need and an important moderator of the impacts of the high psychological demands related to the COVID-19 pandemic. High social support has a protective effect and mitigates the negative impacts of stress on physical and psychological well-being . Thus, tIn addition, there is evidence that CMDs are associated with social inequalities, as individuals in unfavorable socioeconomic conditions  are in a situation of greater vulnerability due to the constant experience of the feeling of insecurity, hopelessness, and risk of violence ,30,31. TSimilarly, higher prevalence of CMD has been observed in people who live alone . OverallEpidemiological indicators, such as the number of new cases, the rates and length of hospital stay, the unfavorable outcomes of the disease, and the number of deaths have shown that the male population has been more impacted, evidencing that being a man is a risk factor for COVID-19 ,34. HoweIn addition to that, population-based studies investigating the mental health situation in the context of the pandemic have included predominantly female samples ,38,39, wGiven the above, it is considered crucial to give visibility to the mental health of male populations and to the aspects associated with the occurrence of mental disorders to strengthen the confrontation of the life dimensions affected by the pandemic in addition to contributing to overcoming this moment of profound health and social crisis generated by the dissemination of COVID-19.Thus, this study aimed at analyzing the relationships between sociodemographic variables, intolerance to uncertainty (INT), social support, and psychological distress, that is, indicators of Common Mental Disorders (CMDs) and perceived stress (PS), in Brazilian men during the COVID-19 pandemic.A cross-sectional study with national coverage, of the web survey type, and carried out during the period of social circulation restriction imposed by the health authorities in Brazil for suppression of the coronavirus and control of the pandemic.The sample was estimated at 923 participants, considering the population of 64,520,660 Brazilian men with Internet access , 50% pre®, Instagram®, WhatsApp®, Grindr®). This is a non-probabilistic sampling technique performed by means of reference chains, where the first eligible and recruited participants invite new participants from their network of contacts  who, in turn, indicate new participants and so on successively, until the estimated sample is minimally reached.The snowball technique  was usedInitially, 25 participants were recruited, five from each of the Brazilian regions, who were called seeds. These participants were encouraged to send the research link to other men from their contact networks. At the end, 27 seeds were recorded, one from each of the Brazilian states.The inclusion criteria adopted were as follows: being digitally literate to access the Internet and being at least 18 years old. The individuals excluded were those non-residents, in Brazil, or who were in the country at the time of data collection.For data collection, a questionnaire that was structured in blocks was used, including the following:(a) Sociodemographic characteristics: sexual identity, age, schooling, self-reported race/skin color, house-sharing or not, work situation, and use of health plans;(b) Common Mental Disorders (CMDs): the Self-Reporting Questionnaire (SRQ-20) was used to screen CMDs. SRQ-20 is validated for use in Brazil with satisfactory performance indicators ,45,46. I(c) Perceived Stress level: the ten-item version of the Perceived Stress Scale (PSS-10) , cross-c(d) Level of intolerance to uncertainty: the Intolerance of Uncertainty Scale (IUS) , 12-item(e) Level of social support: the instrument used was the 2-Way Social Support Scale (2-WSSS) , 20-item®, Instagram®, WhatsApp®, and Grindr® digital social networks by five trained researchers for autonomous and voluntary adherence as well as by making a direct invitation to the individuals who met the eligibility criteria. The data were collected between May and September 2020. The participants had access to the questionnaire after reading and signing the free and informed consent form and thus agreeing to participate in the study. Before each block of questions, the necessary information to answer them were made available as well as the option not to answer any question. The instructions about the recall period for each instrument were clarified.The self-reporting questionnaire was elaborated in a specific data-collection platform and made available through the Internet. From then onwards, it was widely publicized on FacebookConsidering the evidence found in the literature, the understanding of human behaviors related to coping with the coronavirus outbreak and its impacts on people’s mental health, Directed Acyclic Graphs (DAGs) were constructed ,61 to reDAGs are diagrams that allow coding and explaining conceptual hypotheses , with grFor the statistical analysis, the data were transferred from the Google Forms platform to a Microsoft Excel spreadsheet and were later organized in the SPSS software, version 24.0. Initially, a descriptive analysis of the variables of interest was performed for an estimation of frequencies (absolute and relative) and of prevalence values for CMDs. Pearson’s chi-square test was used, with a significance level of 5%, to verify the association between CMDs (outcome) and the independent variables. Subsequently, the database was exported to the Mplus software, version 8.0, for Structural Equation Modeling (SEM) analyses.To perform the SEM, the latent constructs were measured (measuring models) . To assep-value < 0.05. The size of the direct, indirect, and total effects were classified as follows: weak/small (around 0.10), moderate/medium (close to 0.30), and strong/big (>0.50) [To evaluate the structural model constituted by the observed and latent variables, unadjusted and standardized regression coefficients were estimated, with 95% confidence intervals (95%CI) and  (>0.50) .The Weighted Least-Squares Means and Variance Adjusted (WLSMV) estimator was used as a function of the modeling with categorical data. To re-specify the model, the Modification Indices (MI ≥ 10) and the Expected Parameter Changes (EPC ≥ 0.25) were assessed . To asseEthical approval regarding this study was obtained from the institutional ethics committee . All the participants in this study were only included after informed consent had been obtained from them. All procedures performed in this study were compatible with the ethical standards of the institutional research committee and with those of the Declaration of Helsinki and its comparable ethical standards.The study participants were 1006 men. The predominant profile included non-heterosexuals (54.1%), aged from 29 to 39 years old (45.1%), higher education (73.8%), black-skinned individuals (59.2%), without a partner (67.3%), with monthly income up to two minimum wages (41.6%), living with family members/friends (76.6%), and workers (75.0%). A high proportion of people depending exclusively on SUS care and services was observed (41.9%) .The overall prevalence of CMDs was 54.3%. Higher prevalence of CMDs was observed among the youngest individuals (62.7%), those with lower schooling (55.3%), black-skinned (55.1%), without a partner (58.2%), and among those who lived alone (58.3%), did not work (59.4%), had high levels of stress (92.4%), intolerance to uncertainty (82.3%), and received low social support (67.2%). The moderate stress levels reached 60.9%, while 36.0% reported high levels of intolerance to uncertainty. Receiving high social support was mentioned by 36.9% and offering high social support by 39.5% .Socioeconomic status (SES), intolerance to uncertainty (INT), and social support (SS) were treated as latent constructs. In general, the factor loads of the measuring models were high and statistically significant. The factor loads of the SES latent construct were above 0.60. In this construct, the highest load was observed for the occupational situation (OCP) indicator (λ = 0.690), and the lowest was found in the use of health plan (HP) indicator (λ = 0.609) .As for the INT construct, the exploratory factor analysis revealed, based on the eigenvalues, a solution of one predominant factor  with a marked reduction for two factors . In the ESEM, the correlation between prospective and inhibitory IU was 0.992, and only the one-factor solution presented satisfactory fit indices. For this one-factor model, the highest load was observed for the “uncertainty makes me vulnerable” item (I7) (λ = 0.754) and the lowest one for “it is necessary to think about the future to avoid surprises” (I4) (λ = 0.393) .The SS measuring model was initially evaluated by the indicators of the first-order factors: receiving emotional support (RES), receiving instrumental support (RIS), offering emotional support (OES), and offering instrumental support (OIS), all with high factor loads and statistically significant. For RES and RIS, the highest loads were observed for the items “when I’m feeling down, there’s someone I can count on” (S3) (λ = 0.907) and “if I’m in trouble someone will help me” (S13) (λ = 0.845) and the lowest ones for “I feel I have a network of people who value me” (S7) (λ = 0.609) and “there’s someone who can help me fulfill my responsibilities” (S16) (λ = 0.594), respectively. For GES and GIS, greater burdens were verified for “I helped someone with their responsibilities when they were not able to fulfill them” (S18) (λ = 0.700) and “I comfort other people in difficult times” (S11) (λ = 0.867) and lower ones for “I’m a person whom others ask for help with tasks” (S20) (λ = 0.401) and “I’m a person available to listen to others’ problems” (S8) (λ = 0.697), respectively. The high correlation between the constructs of receiving (r = 0.832) and offering (r = 0.864) support endorsed the existence of the respective second-order factors called receiving support (RS) and offering support (OS), without a significant residual correlation (r = 0.430) .The SES, INT, and SS measuring models obtained satisfactory fit indices. The evaluation of the correlations between these latent constructs, both between the second-order factors of the SS model and in the model considering the correlations between all the latent variables, evidenced adequate discriminant validity (r < 0.90) .In the structural model, CMDs was considered as response variable. The SES, RS, OS, and INT latent variables and the self-reported race/color (COL), house-sharing (HS), and perceived stress (PS) observed variables were used as explanatory variables. The direct effects for CMDs were assessed for all the variables of the model, with the exception of OS, and their structural inter-relationships were considered in the paths of specific indirect effects. The estimated structural equation model presented adequate fit indices .p-value < 0.001), SES , HS , PS , and INT  for the CMDs. Thus, black-skinned men, those with higher socioeconomic status, living alone, and with higher levels of stress and intolerance to uncertainty presented higher prevalence values of CMDs. There were strong effects of PS and INT and medium effects of the other variables, showing that high levels of stress and intolerance to uncertainty were the factors with the strongest direct associations with the prevalence of CMDs   .p-value < 0.001), non-black-skinned , who lived alone , received low social support , and offered high social support  had higher stress levels; and higher stress level was associated with a higher prevalence of CMDs. The factors that most contributed (strong effects) to the increase in the level of perceived stress were the following: receiving low social support, living alone, and offering high social support , notably non-black-skinned and those with higher socioeconomic status, had a higher level of intolerance to uncertainty and higher prevalence of CMDs. Intolerance to uncertainty also mediated the effect of perceived stress on CMDs, indicating that men with higher stress levels, especially those with lower socioeconomic status, non-black-skinned, who lived alone and with low levels of receiving social support, and high levels of offering social support, had higher levels of intolerance to uncertainty , resulting in a higher prevalence of CMDs. The factor that most contributed to the increase in intolerance to uncertainty was the high level of perceived stress, with a strong and significant effect , receiving low social support , living alone , high intolerance to uncertainty , and high social support , all strong and significant. There was a small overall effect of socioeconomic status , but the indirect effects, mediated by high levels of perceived stress and intolerance to uncertainty, were robust and significant , with strong and significant magnitude, and offering high social support , of medium size and significant, both mediated by high stress levels. The medium effects of living alone mediated by high stress level , of high stress level mediated by high level of intolerance to uncertainty , and of low social support mediated by high levels of stress and intolerance to uncertainty  also stood out , since our findings identified that black-skinned men, those with higher socioeconomic status, who lived alone, and with higher levels of stress and intolerance to uncertainty had a higher prevalence of CMDs. Thus, an intersectional perspective needs to be employed as a way of explaining the vulnerabilities, inequalities, inequities, and necropolitics that promote overlapping impacts on the health, quality of life, and well-being of men who belong to marginalized groups, an aspect confirmed in our study when we identified the disparity in economic status among non-black-skinned men.Although knowledge about the disease is advanced, there are still many gaps. Commonly, guided behaviors are based on scientific evidence, protocols, and guidelines. However, the recommendations themselves present certain degree of uncertainty, which generates doubts and discomfort in the face of the unknown on the part of authorities, health professionals, and the population . Given tOur study adopted a cross-sectional design and a sampling technique with recruitment by non-probabilistic methods, which limit the causal inference and external validity of the results. In addition to that, participation of men with no access to the interview via the Internet was excluded, which may have over- or under-estimated the prevalence of CMDs in the strata evaluated.The results of this study point out the high prevalence of CMDs in men in the pandemic context and reinforce the need to assess the factors that precede this disease and to replicate the study with women. In addition to that, they endorse the importance of constructing latent variables to measure subjective aspects since three important latent constructs were revealed to be better studied, namely socioeconomic status, intolerance to uncertainty, and social support.In addition to that, the study allowed identifying a higher prevalence of CMDs among younger men, with lower schooling, black-skinned, without a partner, who lived alone, did not work, with high levels of stress and intolerance to uncertainty, and who received low social support. It was also evidenced that the high levels of stress and intolerance to uncertainty were the factors that presented the strongest direct associations with the occurrence of CMDs.These findings indicate that stress and intolerance to uncertainty are important factors in understanding mental suffering among men. Dealing with unexpected situations, with the loss of close people and future life perspectives accentuates stress levels, with the consequence of the occurrence and/or worsening of CMD. This situation is even worse in the presence of receiving low social support from close people and/or the government, as this support has an important mitigating effect on mental suffering, being able to resolve the harmful impacts of high psychological demand and the low control over adverse situations.Thus, the importance of developing mental health promotion policies and actions for male populations is highlighted, considering that intervening in the aspects associated with the occurrence of CMDs is crucial to facing the impacts of the COVID-19 pandemic in the different life dimensions.


In general, the severity of this nanomaterial's toxic effects varies according to the administration route, the dose to be administered, the method of GO synthesis, and its physicochemical properties. This review brings together studies on the method of synthesis and structure of GO, characterization techniques, and physicochemical properties. Also, we rely on the toxicity of GO in cellular models and biological systems. Moreover, we mention the general mechanism of its toxicity.Nanomaterials have been widely used in many fields in the last decades, including electronics, biomedicine, cosmetics, food processing, buildings, and aeronautics. The application of these nanomaterials in the medical field could improve diagnosis, treatment, and prevention techniques. Graphene oxide (GO), an oxidized derivative of graphene, is currently used in biotechnology and medicine for cancer treatment, drug delivery, and cellular imaging. Also, GO is characterized by various physicochemical properties, including nanoscale size, high surface area, and electrical charge. However, the toxic effect of GO on living cells and organs is a limiting factor that limits its use in the medical field. Recently, numerous studies have evaluated the biocompatibility and toxicity of GO  The toxic effects of GO depend on several factors, including the route of administration, the dose to be administered, the method of synthesis of GO, and its physicochemical properties. These factors influence and increase the complexity of comparisons between different studies on the toxicity of GO.Nanoparticles are widely used in electronics, aeronautics, energy, agriculture, cosmetics, medicine, textile production, and many other fields. They are currently used to administer drugs, proteins, genes, vaccines, polypeptides, and nucleic acids . AccordiIn this review, we have introduced the synthesis methods, structure, characterization techniques, and GO properties. In addition, we presented and discussed available toxicological studies of GO in vitro and in vivo. We summarized the cytotoxicity of GO in cellular models. We then focus on the pathways by which GO enters the body and the role of biological barriers. We also present the biodistribution, biotransformation, and excretion of this nanomaterial and also discuss the toxicity of GO in different body systems. Finally, we showed the general mechanism of toxicity, to better understand the toxic effects related to the exposure of GO to improve the biological safety of this nanomaterial and facilitate its use in the biomedical field.3) is fuming nitric acid [The first synthesis of GO is often attributed to Brodie. In 1859, British researcher Benjamin Brodie carried out research that mainly consisted of oxidizing graphite sheets using potassium chloride  was added to get a higher oxidation degree and to eliminate manganese from the dispersion  and increased KMnO4. This method's advantage is the absence of generation of toxic gases, such as NO2, N2O4, or ClO2, in the reaction, easy temperature control, and gives GO powders a higher degree of oxidation.The Tour group proposed improving the Hummers method at the University of Rice in 2010 . They ha4 between the graphitic layers produces another spontaneous expansion that resembles foam of graphitic material. The reaction takes place in the middle of sulfuric acid.In 2013, Sun and Fugetsu of Hokkaido University  introduc2FeO4) as a strong oxidant. In this method, a mixture of graphite powder and K2FeO4 dispersed in concentrated sulfuric acid was loaded into a reactor and stirred for 1 hour at room temperature. The product was washed with water by repeated centrifugation to obtain highly water-soluble GO.Two years later, Peng and his collaborators  proposed4 and in the presence of concentrated sulfuric acid, resulting in the formation of pasty slurry. The second step requires the exfoliation of the graphite. Indeed, the production of GO is limited by temperature and only occurs when the water bath is at about 30°C. Hydrolysis at 90°C for 1 hour completes the third step. Purification of the product is carried out by centrifugation with hot water until the dispersion is neutral in the fourth step. This method's advantage is related to the improved operational safety conditions and the production of a type of GO that contains less than about 20-30% oxygen domains. This type of GO can be used for filtering/remediation or biosystems due to the common toxic effect.Pendolino and his collaborators have improved another procedure called the 4-step method . It consIn all of the above synthesis methods for preparing GO, certain limitations are encountered. The use of sodium nitrate or potassium chlorate in the Brodie or Staudenmaier methods leads to explosive results. In contrast, sodium nitrate (Hummers) or fuming nitric acid introduces heteroatoms or defects on the GO structure that affects reactivity . Anotherμm and the largest size from 256 μm) using a swelling crystal strategy using oxidation-monolithic crystal swelling that can ultimately convert graphite into the ultrawide GO [Recently, a more innovative study of the synthesis of the GO has been proposed. In this study, researchers prepared the ultrawide GO , scanning electron microscopy (SEM), transmission electron microscopy (TEM), Raman spectroscopy, solid state nuclear magnetic resonance (Ss-NMR), Fourier transform infrared spectroscopy (FT-IR), X-ray induced photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and thermogravimetric analysis (TGA).μm [Atomic force microscopy is a technique used in nanotechnology to study materials at the nanoscale. It is capable of characterizing the lateral size and thickness of the GO layer. In general, the height profile reveals a diameter of 1–1.2 nm. Simultaneously, the lateral size can be in the order of tens to hundreds of micrometers, depending on the synthesis and postsynthesis processing, e.g., sonication. Analyses obtained by this technique have shown that the GO layers' thickness is about 1.1 nm and a lateral size ranging from 500 nm to 50 μm .μm  is attributed to the disorder's crystal structure. This fact correlates G-band to carbon sp2 and D-band to the presence of sp3 and thus to oxygen domains [Raman spectroscopy is based on the phenomenon of inelastic scattering of a monochromatic light beam . This te domains , 46.2 hybridized carbon materials. The vibration band G (around 1580 cm−1) represents the atoms' vibrations in the plane and corresponds to a permanently active mode.The Raman analysis was presented in −1) corresponds to a vibration mode called breathing, which does not obey the selection rules that determine whether a mode is active.The D band (around 1335 cm−1) is a harmonic of the D-band and provides information on the graphene planes' stacking order. In monolayer graphene, the 2D band is an intense Lorentzian peak (about four times the intensity of band G). The shape of the 2D strip then changes according to the number of planes, then from five planes of thickness, becomes identical to that of hexagonal graphite.The 2D band (around 2700 cmμm (The use of pulsed solid-state NMR on carbon-13 (PD) with rotation at the magic angle (12.5 kHz) makes it possible to identify the GO structure's different carbon atoms characteristic. μm  is twiceμm . Small Oμm) of frequencies between 4000 and 200 cm−1 [−1 is attributed to the stretching vibrations of the O-H group. The absorption peak at 1730 cm−1 and 1630 cm−1 can be attributed to the C=O stretching of the carboxyl and carbonyl parts' functional groups. The two absorption peaks at about 1226 cm−1 and 1044 cm−1 are attributed to the C-O group's stretching vibrations [Fourier transform infrared spectroscopy is a technique for studying chemical bonds' movement by measuring the absorption of electromagnetic radiation from a compound. This analysis is based on the excitation of molecular bonds in a sample by infrared radiation , during the pyrolysis of the carbon skeleton.2g−1) [GO is characterized by the diversity of its physicochemical properties. The main characteristic is that it behaves like a hydrophilic material, thanks to the hydroxyl groups, epoxides, ketones, and carboxylic acids. The presence of these oxygenated groups considerably modifies the properties of GO. They allow biochemical and bioconjugation reactions to occur at the basal plane and the edges of the GO . These r2g−1)  and mech2g−1) , and it 2g−1) . Studies2g−1) . Particl2g−1) . This ma2 hybridization, protein adsorption on the GO's surface occurs by hydrophobic-hydrophobic interaction [π on the basal plane of the GO surface, the stacking interactions π-π may also occur [GO has a high adsorption capacity for proteins and antibodies. Proteins adsorbed in GO have been shown to increase protection against proteolysis . The meceraction , causingeraction . Besideseraction . Still, eraction . Howevereraction . On the eraction . Finallyso occur .GO is characterized by the diversity of its physicochemical properties. It is hydrophilic, biocompatible, has a high specific surface, a mechanical resistance, and a semiconductor nanomaterial because of its oxidation degree. On the other hand, GO has a high adsorption capacity for proteins and antibodies, and it is considered superb catalyst support.The toxicity of GO in cells is due to several factors, including dose, lateral size, and surface charge , 73. To in vitro is closely related to incubation conditions, including exposure dose, culture time, incubation temperature, and cell type [μg/mL did not exhibit toxicity to human fibroblast cells, and the dose of more than 50 μg/mL exhibits cytotoxicity such as decreasing cell adhesion, inducing cell apoptosis, and entering into lysosomes, mitochondrion, endoplasm, and the cell nucleus [On the contrary, there are several studies on the toxic effects of GO on cells. Researchers have reported that incubation of human breast cancer cells MDA-MB-231 with GO caused a decrease in cell viability due to the dose of GO exposure  Table 1Table 1. ell type . Also, t nucleus .μg/mL showed no cytotoxicity on A549 cells [μg/mL concentration of GO, observing a dose- and time-dependent reduction in viability at higher concentrations [Similarly, the dose of 20 49 cells . The met49 cells . The deptrations .μm and 350 nm have very different lateral dimensions but also contribute to the amount of absorption in macrophages. Similar amounts of antibody opsonization and active Fcγ receptor-mediated phagocytosis have been shown to cause this behavior. While the microdimensional GO showed different intracellular locations and induced much stronger inflammatory responses.The lateral size of the GO also affects cytotoxicity. Researchers have shown that cytotoxicity depends on the lateral size and density of the functional groups of the GO. They found this result from exposure of human lung cells (BEAS-2B) and alveolar epithelial cells (A549) to three types of GOs that differ in lateral size and functional group density. They also found that GO and thermally reduced GO are more toxic than chemically reduced GO . AnotherAnother critical factor inducing cytotoxicity is the surface charge of the GO. Studies have suggested that GO also has an impact on cell internalization and absorption . The intin vitro toxicity of GO in the literature, it can be said that this nanomaterial can be either harmless or toxic to cells. The degree of toxicity is a function of the physicochemical properties of GO and the experimental conditions.Recently, a new study evaluated the toxicity of GO in the rat cardiomyoblast H9c2 cell line. It demonstrated that GO induced cardiotoxicity, mitochondrial disruption, generation of reactive oxygen species, and DNA interactions . Based oThe natural routes of entry of nanoparticles into an organism are inhalation, ingestion, and dermal. As a result, some toxicological studies in animal models mimic this natural contamination pattern by directly bringing nanoparticles into contact with organisms. Others choose to administer GO by intravenous, intraperitoneal, and subcutaneous injections . These iGO is considered to be an excellent drug delivery system . It is uOn the other hand, studies have suggested that intestinal absorption of nanoparticles is limited after oral administration, and their excretion is rapid . Other sMoreover, GO-based nanomaterials have been structured as topical antimicrobial media in the form of bandages, ointments , or cottOther biological barriers are also mentioned in the literature. Studies have shown that GO particles with a 54.9 ± 23.1 nm diameter had difficulty penetrating the hemato-testicular and hemato-epididymal barriers after intra-abdominal injection. Also, the sperm quality of the mice was not affected, even at a dose of 300 mg/kg . RegardiThe biodistribution, biotransformation, and excretion of GO can be influenced by several factors, including routes of administration, physicochemical properties, particle agglomeration, and surface coating. Zhang et al.  found thGO can undergo significant biotransformation and modify its physicochemical properties due to its greater chemical reactivity . Qi and In vivo experiments in mice confirmed morphological alterations of the GO in a realistic lung microenvironment. These results suggested that the biotransformation of GO may significantly alter their inherent properties and thus affect their biosafety [In contrast, ALF changes enhanced the adhesion of large sheet-like GO aggregates to the plasma membrane without cell uptake . Other siosafety .GO excretion varies in different organs. In the lungs, GO is challenging to eliminate, causing inflammation, cell infiltration, granuloma formation, and pulmonary edema , 105. InIn contrast, small particles of about 8 nm can enter the renal tubules in the urine and be rapidly removed without any toxicity . The rouIn order to examine the pulmonary toxicity of GO, studies have used a single 6-hour inhalation of GO at low and high concentrations in rats. After this exposure, the animals were allowed to recover for 1 day, 7 days, or 14 days. The results of this exposure showed that the levels of microalbumin and lactate dehydrogenase in the bronchoalveolar lavage (BAL) fluid were not significantly altered. Similarly, the total number of macrophages, leukocytes, and lymphocytes was not significantly altered in the BAL fluid. Moreover, histopathological analyses of rat lungs showed the only GO absorption in alveolar macrophages in the high concentration group . Based oOn the other hand, intratracheal instillation of GO in vivo resulted in pulmonary toxicity. Li et al.  found thOral gavage experiments in animals found that GO was not absorbed from the gastrointestinal tract . Fu and Work has shown that GO is a nephrotoxic product and that its toxicity can be mediated by oxidative stress . These sFurthermore, histological analyses did not reveal any toxicity in the renal tissue. These results indicate that the injected GO nanoparticles do not have a toxic impact on the mice after 4 weeks of injection. In parallel, Jasim et al. observed significant urinary excretion after intravenous administration of GO to mice . They obCaenorhabditis elegans [Caenorhabditis elegans.GO and graphene-based nanomaterials have been widely used in recent years in biomedical applications to treat brain tumors, intracranial and spinal biocompatible devices, and biosensing and bioimaging techniques. However, the potential health risk and neurotoxic potential of GO are not yet clear. Amrollahi et al.  evaluate elegans . Indeed,On the other hand, Rauti and his colleagues  have proStudies have shown that GO and rGO are capable of causing damage to zebrafish embryos. Exposure to different concentrations of these nanomaterials influenced the hatching rate and body length of embryos. However, no malformations or mortalities were observed in zebrafish embryos after exposure to these two nanomaterials . Anotherα-glucosidase, lactate dehydrogenase, glutathione peroxidase, and acid phosphatase, were not affected by GO treatment [Further work has shown that male mice given high doses of GO (25 mg/kg mice) by intravenous injection exhibited normal sex hormone secretion and maintained regular reproductive activity. All untreated females mated with male mice intoxicated with GO were able to produce healthy offspring. Histological analyses of the testes and epididymis with the activities of several epididymal enzymes, including reatment . On the reatment .μg/mL altered gene expression. Furthermore, they showed that intravenous injection of GO at 4 mg/kg for 5 days in mice induced the formation of micronucleated polychromatic erythrocytes [Studies have shown that GO is capable of inducing genotoxicity. Liu and his colleagues found that GO induced mutagenesis at the molecular level. The use of GO at concentrations of 10 and 100 hrocytes . Anotherhrocytes .μg/mL) [On the other hand, another study showed that GO did not induce significant genotoxicity in FE1 murine pulmonary epithelial cells even at relatively high doses (5-200 μg/mL) . Therefoμg/mL) .The effects caused by carbon nanoparticles, including GO, can be highly dependent on the organisms and biomarkers considered. The mechanism of toxicity of GO is explained as follows:E. coli bacteria and caused the release of intracellular components, leading to cell death [Studies have shown that the interaction between GO and cell membranes is one of the main causes of GO cytotoxicity . Direct ll death . Anotherll death . In addill death .Euglena gracilis with GO for ten days caused growth inhibition, decreased photosynthetic pigments, and increased ROS levels [The toxicity of GO nanosheets is often manifested by the production of reactive oxygen species (ROS), leading to oxidative stress characterized by an imbalance between free radicals and antioxidants. ROSs act as secondary messengers in many intracellular signaling cascades and lead to cellular macromolecular damage, such as degradation of membrane lipids, DNA fragmentation, protein denaturation, and mitochondrial dysfunctions . In a stS levels . TherefoS levels . The accS levels .On the other hand, small GO could be degraded by lysosomes and eliminated from the body without causing observable toxicity. On the reverse, large GOs could cause damage to the cell membrane by binding to proteins and interacting with phosphatidylcholine, leading to ROS production, and increasing the dose and duration of exposure to GO results in a progressive decrease in the activity of SOD and GSH . These oIn this review, we have given a detailed overview of the synthesis methods of GO, its structure, different characterization techniques, and its physicochemical properties. Through characterization techniques such as SEM, TEM, and XRD, it has been demonstrated that GO has a nanoscale size. Due to its small size and physicochemical properties, GO is used in several applications, especially biomedical ones. Although GO is useful for many applications, there is still a risk related to its “toxicity,” limiting its uses. Studies conducted so far indicate that the toxicity of GO could depend on its size, synthesis methods, route of administration, and exposure time. In addition, we presented the different toxic effects of this nanomaterial at the cellular and systemic level of the body with discussions on the underlying toxicological mechanism. We also highlighted the role of biological barriers to the entry of GO into the body and its toxicokinetics. ROS-mediated cellular damage has been postulated as a primary mechanism of GO cytotoxicity. In general, available GO toxicity studies are mainly limited to evaluating acute toxicity, while chronic toxicological studies lack. However, the routes of administration, the dose to be administered, and the physicochemical properties directly influence the toxicity of GO. The analysis of these factors allows determining its toxicity. To better understand the toxicological mechanism of this nanoparticle, it is necessary to identify the molecular targets involved in the toxicity and evaluate the benefits and risks of GO for health to benefit from the advantages of nanotechnologies to minimize the risks for human health.


Then targeted antifungal treatment was selected quickly and his temperature gradually returned to normal. Thus, we report the case in which mNGS was an auxiliary method to diagnose mucormycosis, and discuss this case in combination with relevant literature, in order to improve the clinical cognition of this technology.mNGS(metagenomics Next Generation Sequencing), as a novel culture-independent approach, demonstrated the capability of rapid, sensitive, and accurate pathogen identification. At present, there have been many case reports about the use of mNGS to assist in the diagnosis of bacterial, fungal, viral and parasitic infections and to guide clinicians to determine appropriate treatment. However, the clinical understanding of this technique is not comprehensive, and the experience of using it is relatively limited. We reported a 53-year-old man who was admitted to hospital with a high fever and headache. His inflammatory biomarkers were markedly elevated. Based on the clinical presentation, He was initially diagnosed as having an intracranial infection of unknown etiology and received empirical antibiotics and systemic supportive treatment. But these did not relieve his symptoms. Both the blood and CSF specimens were examined using traditional culture, serological testing, and mNGS. Traditional culture and serological testing produced negative results, while the mNGS revealed the presence of a potential pathogen,  Mucormycosis, also known as zygomycosis, is a highly invasive conditional fungal infection. It has low incidence, about 1.2 per 1 million people per year  is the most common type, accounting for about 75% of reported cases  is a new genomics-based pathogen detection technology, which has the advantages of high detection rate and fast detection speed. Thus, we report the case of intracranial infection caused by A 53-year-old man presented to our hospital with a 7-day history of fever and severe headache and a 1-day history of left extension of the tongue and slurred speech.11 days before arriving at our hospital, the patient developed periodontal pain and maxillofacial swelling without obvious inducement and received antibiotics in the local hospital. However, his symptoms were not significantly relieved, neither did his body temperature drop.On admission, vital signs were shown as: body temperature was 37.2°C, respiration was 20 beats per minute, heart rate was 84 beats per minute, blood pressure was 158/81 mmHg. Physical examination revealed a 1 cm lesion near the gum of the patient's upper incisor; the right side of the light reflection disappeared and the left side is dull; the left nasolabial groove is shallow and the patient has a left extension of the tongue. Blood routine examination (BRE) showed that white blood cells were 27.62 × 109/L (neutrophil accounted for 89.1%). The procalcitonin (PCT) level was elevated to 0.120 ng/mL, C-reactive protein (CRP) reached 178.30 mg/L. Magnetic resonance imaging (MRI) showed a lesion in the brainstem. Hence, based on previous clinical experience, the patient was diagnosed preliminarily with brainstem encephalitis and purulent gingivitis. An initial treatment of meropenem (2g/8h iv) and linezolid (0.6g/12 h iv) had been empirically administrated. At the same time, enteral nutrition, reducing intracranial pressure, anti-heart failure, organ function protection, analgesia and other symptomatic treatment were given. But there was no significant relief of his symptoms.20;cytology showed 46 × 106 leukocyte/L, glucose: 4.05mmol/L, chloride: 125.3 mmol/L, lactic acid: 2.90 mmol/L and there was no evidence of fungal, or acid-fast bacilli infection. Serological testing was performed, including 1,3-β-D glucan test (G test), galactomannan antigen test (GM test) and bacterial smear test; however they were all negative. Computerized tomography (CT) showed paranasal sinusitis and bilateral hypertrophic inferior turbinate. Consider the possibility of infection by a particular pathogen, empirical antifungal and antiviral therapy of ganciclovir intravenous (0.5g/12h iv) and voriconazole (400mg/12h iv) was given.In order to improve the etiological examination, both his peripheral blood and CSF specimens were examined to investigate possible etiology using traditional culture and mNGS. The CSF presented a colorless and transparent appearance, intracranial pressure was 600 mmHLichtheimia ramosa  but he suffered developed brainstem failure. We use high doses of vasoactive drugs and still have difficulty maintaining his blood pressure. After communicating with the patient's family, they requested to be discharged.mucor in a patient with intracranial infection. This patient was admitted to hospital due to high fever and headache. Traditional culture and serological testing did not determine possible infection etiology, while mNGS identified mucor in the CSF specimen. Combined with his clinical characteristics, his diagnosis was confirmed to be a ROCM.Here, we reported a case in which mNGS helped clinicians rapidly and accurately identifying mucormycosis occurred in patients with “no underlying disease” , (M test), , 3 – β-DM test), , 3 – β-DImaging examination: Imaging examination is helpful for the detection of mucosal edema and thickening, extraocular muscle and soft tissue swelling, bone destruction, brain parenchymal lesions, intracranial vascular occlusion and other conditions in the affected areas before the appearance of clinical symptoms, and can also be used to judge the therapeutic effect and determine surgical methods. Patient's CT showed paranasal sinusitis and bilateral hypertrophic inferior turbinate and his MRI showed multiple intracranial soft tissue infections, suppurative otitis media, and gingival infection. All of his imaging findings are not typical and have poor clinical suggestive effect.Molecular biological examination: The commonly used clinical molecular diagnostic method is PCR, which is more sensitive than pathogen culture  The susceptible people should consider this disease when they have symptoms such as fever, headache, swelling of maxillofacial region and nasal congestion and should carry on the etiology examination and the tissue biopsy pathology examination in time to achieve early diagnosis and treatment. (2) The discovery of mucor's characteristic by biopsy is the gold standard for diagnosis of this disease, but medication can be taken as soon as possible according to clinical experience, so as not to delay diagnosis and treatment. (3) For patients with intracranial infection with atypical symptoms and rapid disease progression, the pathogen must be identified as soon as possible, and mNGS has a certain auxiliary role in diagnosis.In this case, the early medication was not targeted and did not successfully prevent the progression of the disease. It was not until the mNGS in the CSF indicated the infection of In conclusion, specific pathogens should be highly suspected based on the patient's underlying disease history, clinical manifestations, and ineffective use of conventional antibiotics. However, due to the diversity of pathogens, the limitations of traditional detection methods, and the uneven judgment of clinicians on target pathogens, many infectious diseases can not be identified in clinical practice. Traditional etiological detection methods are time-consuming and have low sensitivity, which cannot meet the needs of early diagnosis and treatment of this kind of disease. Sensitive, rapid, accurate and simple diagnostic techniques are needed. In recent years, the emerging mNGS technology has helped to improve the level of diagnosis, and has played a role in the detection of a variety of pathogens. While the technique is currently used only as a second-line option in cases where testing for other pathogens is inconclusive, but with the rapid advances in genomics and sequencing, mNGS will be more and more widely used in the traceability, detection, typing and drug resistance evaluation of infectious diseases, and will develop rapidly in the direction of fast and economic development.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.The studies involving human participants were reviewed and approved by First Affiliated Hospital of Zhengzhou University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.YCL, JZ, and YGL analyzed and interpreted patient data. BH, LJD, and ZYS performed the experiments. YCL, CCW, and MX analyzed the genomics data. YCL and BH wrote the manuscript. All authors read and approved the final manuscript.This research was equally funded and supported by Henan Province Science and Technology Project Grant 192102310054. Fund of the First Affiliated Hospital of Zhengzhou University 2018020136.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.


This study explored the correlation between mental toughness (MT) and physical activity (PA), and the moderation role between PA intention and subsequent behavior among college students and wage earners. Five hundred ninety-one college students  aged from 19 to 24 and 285  wage earners aged from 27 to 58 recruited from seven colleges and five cities in China. A Theory of Planned Behavior (TPB) questionnaire, MT Inventory, and the International PA Questionnaire was completed online. Results showed that attitudes, subjective norms, and perceived behavioral control explained 46.5 and 38.3% variance in PA intention among college students and wage earners separately. Intention predicted PA behavior significantly among college students and wage earners. Structural equation modeling indicated that the TPB model and the moderation model have an adequate to good fit except the TPB model among wage earners. MT was positively correlated with PA among college students and wage earners and had a significant moderation role in intention-behavior gap among college students and partially affected the transfer of PA intention to behavior among college students. Individuals with high MT had high levels of PA regardless of intention, while PA of those with low MT was low and unstable. Future research should further explore the correlation between intention and PA and the moderation role of MT in different populations using a longitudinal study in order to better understand the correlation between intention and PA, and the transition from intention to PA and better guidance PA intervention to promote PA. Physical activity (PA) refers to any type of PA with energy consumption due to skeletal muscle contraction, such as daily work, housework, physical exercise, and activities for entertainment . The heaIn China, according to the 2019 General Administration of Sport survey, only 33.9% of adults participate in PA  in ChinaTPB is important in the explanation of health-related behaviors such as PA behavior, which considers that “intention” is the primary determinant of health-related behaviors . Origina•Attitudes reflect an individual’s cognition and evaluation of the behavior, which could be positive or negative.•SN emanates from influential individuals, especially family members, leading to social pressure.•PBC is a concept similar to self-efficacy, which refers to an individual’s belief in his or her own behavioral ability.Many international studies , as wellAs a result, researchers began to focus on variables which moderated the PA intention-behavior gap, e.g., self-efficacy, extraversion, habit , self-reMental toughness refers to an individual’s capacity to cope with difficult life circumstances  and is cGiven that many individuals with PA intentions are still inactive, a high level of PA among individuals may be due to the smaller intention-behavior gap being reduced by high MT. Two studies explored the moderation role of MT in the intention-behavior gap . One stuPhysical activity intention and behavior may vary according to the differences in age and social identity. In a recent study, there was no significant difference in the MT of students with high PA compared with those already in the workforce (wage earners), and the positive correlation between MT and PA was only found in students, not in wage earners . CompareThe purpose of this study was to explore the correlation of TPB constructs, MT, and PA behavior among college students and wage earners in China. The study first analyzed the correlation of attitudes, PBC, SN, PA intention, and behavior and hypothesized that attitudes, PBC, and SN can significantly predict PA intention, and intention could also predict PA. Second, we analyzed the moderation role of MT in the PA intention-behavior gap among the two separate groups. We hypothesized that MT correlated with PA in both college students and wage earners and was a critical moderator which influenced the transition of PA intention to behavior.Ethical approval was first obtained from the Human Research Committee of Tianjin University of Sport. A sample of 591 college students  aged from 19 to 24 and 285 wage earners  aged from 27 to 58 was recruited from seven colleges and five cities in China based on a convenience sample principle. Students from physical education and sports training were excluded from this study.The study adopted a prospective design with data being collected in two steps at a 7-day interval. An online version of all questionnaires was designed by the first author. All participants volunteered for this study and joined a WeChat group in order to collect the data of PA. All participants were asked to undertake informed instruction before data collection and then complete the questionnaires.First, participants finished the questionnaires online, including demographic information, such as age, sex, height, and weight, TPB questionnaire, and the MT Inventory. One week later, all participants received a message that asked them to scan the WeChat QR code to complete the International PA Questionnaire. Twelve students and nine wage earners were excluded from further analysis due to lack of data.Participants completed the Chinese version of TPB questionnaires including four constructs: intention, attitudes, subject norms, and PBC . Intenti2/df = 1.84, CFI = 0.998, TLI = 0.995, RMSEA = 0.046, SRMR = 0.005. The internal consistency coefficient of the MTI is 0.981.The Mental Toughness Inventory (MTI)  is a selThe brief edition of the International Physical Activity Questionnaire (IPAQ-Chinese) is a self-report questionnaire used to measure walking, moderate PA, and vigorous PA at work and home and for leisure in 7 days. The volume of total PA is computed in METs—min/week—and converted to MET minutes through weighting each kind of activity by its energy requirements by using formula “physical activity = 3.3 × cumulative low intensity activity time per week + 4.0 × cumulative medium intensity activity time per week + 8.0 × cumulative high intensity activity time per week.” The PA questionnaire has been internationally validated as a reliable assessment of PA for adults aged 18–65 .All variables were presented as mean and standard deviation. Pearson analysis was used to analyze the correlation of attitudes, PBC, SN, PA intention, and behavior. A confirmatory factor analysis was conducted using Mplus 6.0 to test the factorial validity of the MT questionnaire. We also conducted structural equation modeling using Amos 22.0 to examine whether the model from the TPB constructs to PA fit the data, based on the standard for assessing adequate model fit. CFI and TLI > 0.90, RMSEA and SRMR < 0.08 . More stF = 84.703, p < 0.001]. Attitudes  was the strongest predictor of intention, followed by PBC , while SN  was not a significant predictor.Simultaneous multiple regression control of age, gender, and BMI showed that the TPB predictors explained 46.5% variance in PA intention among college students , and the contribution of attitudes, SN, and PBC was different to that of college students. Attitudes  and SN  were strong predictors of intention, while PBC  was not a significant predictor among wage earners.The TPB predictors also explained 38.3% of the variance in PA intention among wage earners , 95% CI ), and 9.6% of the variance of PA among wage earners , 95% CI ). In addition, the moderation model explained a substantial portion of variance (16.37%) of PA among college students  and 14.09% variance of PA among wage earners, . It appeared that MT made little contribution to PA among wage earners .Correlations between intention, MT, and PA that were entered into the moderated regression model are presented in t = 2.684, p < 0.01, 95% CI ), but not significant at moderate levels  or high  of MT among college students , moderate , and high .Based on the suggestion proposed by students , and alsstudents , with lointention is the primary determinant for health-related behavior (Based on the TPB, behavior . HoweverThe TPB constructs could be relevant in explaining the variance of PA intentions, with 46.5% among college students and 38.3% among wage earners, respectively, which was consistent with previous studies . This stPBC could significantly predict behavioral intention among wage earners. This was partially consistent with previous results . StudiesMental toughness positively correlated with PA both among college students and wage earners. The results further supported the view that MT was a determinant of PA . PreviouExcellent executive control and self-monitoring abilities  of high Intention can significantly predict PA among college students and wage earners. When MT was added to the intention-behavior gap as a moderation variable, its contribution in predicting PA increased, from 7.4% of the variance to 16.37% among college students, and 9.6% of the variance to 14.09% among wage earners. Previous studies found that MT could give the individual a perception of strength, and improved their behavioral motivation level , and espIndividuals with high MT believe in their ability to participate in PA and have the determination and commitment to actively pursue and achieve PA goals . TherefoFurther analysis using the simple slopes showed that PA of low-mental-toughness individuals was more susceptible to PA intention, while PA of both college students and wage earners with high MT was scarcely influenced by intention, suggesting that individuals with high MT undertook high levels of PA regardless of intention. The result was inconsistent with previous studies. First, cultural differences may be a possible consideration. Individuals in Western countries may have a more positive PA habit and often participate in PA , which cSecond are differences in the characteristics of the samples. The samples in Third, the difference of the TPB questionnaire applied in In conclusion, TPB constructs could be significant in the prediction of PA intention both among college students and wage earners. MT is an important and positive predictor of PA, which suggests that researchers should consider the role of MT in intervention in the PA of adolescents. MT intervention may be an effective strategy to promote PA. While intention directly affects PA, its contribution to behavior decreases once MT is added to the mode: MT is an important variable that influences the transition of intention to PA. Individuals with high MT display high levels of PA regardless of intention, while the PA of those with low MT remains low and unstable. Future research should further explore the correlation between intention and PA and the moderation role of MT among different populations using a longitudinal study to better understand the intention-behavior gap.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Human Research Committee of Tianjin University of Sport. The ethics committee waived the requirement of written informed consent for participation.YY and ZC contributed equally to this work. Both of them designed this study, oversaw data collection and data analysis. YY and ZH contributed to data interpretation and the drafting and revision of the manuscript. WD was also responsible for data collection. 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.


Leishmania infantum produces an endemic disease in the Mediterranean Basin that affects humans and domestic and wild mammals, which can act as reservoir or minor host. In this study, we analyzed the presence of the parasite in wild American minks, an invasive species in Spain. We screened for L. infantum DNA by PCR using five primer pairs: Two targeting kinetoplast DNA (kDNA), and the rest targeting the ITS1 region, the small subunit of ribosomal RNA (SSU) and a repetitive sequence (Repeat region). The detection limit was determined for each method using a strain of L. infantum and a bone marrow sample from an infected dog. PCR approaches employing the Repeat region and kDNA (RV1/RV2 primers) showed higher sensitivity than the other PCR methods when control samples were employed. However, only PCR of the Repeat region and nested PCR of SSU (LnSSU) detected the parasite in the samples, while the other three were unable to do so. The majority of the analyzed animals (90.1%) tested positive. American mink may act as an incidental host of the disease for other mammals and should be further investigated, not only for their negative impact on the local fauna, but also as carriers of zoonotic diseases. Neovison vison) is a mustelid native of North America that was introduced in Europe for fur farming. Throughout recent decades, accidental or deliberate escape of mink from farms caused the establishment of stable feral populations. In Spain, the first observation of feral animals was detected at the end of the 1970s, and at present, there are stable populations distributed throughout the northwest and east of the Iberian Peninsula  [Lutra lutra) or the endangered European mink (Mustela lutreola) [Toxoplasma gondii [The American mink (utreola) . For thiutreola) . Moreoveutreola) , protozoa gondii , and vira gondii  and SARSLeishmania infantum is one of the etiological agents of zoonotic leishmaniasis, which is an endemic parasite of the Mediterranean Basin. It is mainly transmitted by phlebotomine sandfly bites [Leishmania parasites [ly bites , althougarasites . The disarasites .Occasionally, conditions that favor the expansion of the disease, including spatial and temporal coincidence of humans, vectors, and potential reservoirs appear, lead to human outbreaks. The ideal habitats for vector expansions are warm areas rich in organic material, such as caves or burrows where animals breed, including wild mammals .Leishmania in different species of wildlife, including wolfs, foxes, mustelids, bats, hedgehogs, and rodents [L. infantum has been reported in mink farms in Greece [L. infantum.When leishmaniasis outbreaks appear, an exhaustive search of potential reservoirs and vectors begins, and monitoring activities can reveal new hosts for the parasite. In the case of Spain, an outbreak starting in 2009 in Madrid highlighted the importance of wildlife as potential reservoirs of the disease, specifically lagomorphs . Thencef rodents ,16,17,18 rodents . The Amen Greece  and in oL. infantum by PCR using five primer pairs to detect the DNA of the parasite.In this article, we explored the role of wild communities of American mink as a host of Neovison vison, according to the status in each country. One of the objectives of the LIFE13 NAT/ES/001171 and the Spanish strategy for control and eradication of the American mink is to capture and euthanize as many animals as possible. The total number of animals captured in the Valencian community in previous control programs was 220 in 8–10 years, which means that the present sample approaches the highest number of animals possibly subject to eradication per year, although a precise number of animals in the area is not available. More information on the distribution of stable populations of feral American minks in Spain can be found elsewhere [Spleen samples from American minks of three different river basins  located in the Valencian Community (Spain) were analyzed . The anilsewhere . The anilsewhere . BiometrL. infantum generously supplied by Dr. J.M. Requena was used to calculate the detection limit of the PCR protocols employed. The isolate was grown in RPMI containing 15% fetal bovine serum  and 10 µg/mL of hemin . In the exponential phase of the culture (day 3 post-inoculation), promastigotes were counted in a Neubauer chamber using 1:1 Trypan blue (4%) in 10% formaldehyde. One million promastigotes were employed for DNA isolation. The parasites were recovered by centrifugation at 800× g for 5 min. The sediment was washed three times with phosphate-buffered saline (PBS) solution, followed by centrifugation in the same manner. We isolated DNA from the pellets obtained using a commercial DNA extraction kit , following the manufacturer’s protocol. Bound DNA was eluted in 50 µL of elution buffer and stored at −20 °C until use. A culture of the strain “JPC” (MCAN/ES/98/LLM-722) of −7-fold dilutions, using DNA from the L. infantum culture. The initial DNA concentration was quantified by spectrophotometry (260/280 nm ratio) using a basic Eppendorf BioSpectrometer® , and 20 µg/mL was obtained.The limit of detection was evaluated by conventional PCR, using four different targets  by 1/10 serial dilutions, up to 10L. infantum, kindly supplied by Prof. Luis Cardoso , was used. Again, 1/10 serial dilutions, up to 10−7-fold dilutions, were employed to compared the five PCR protocols. To evaluate the detection limit of each PCR protocol, and also as a positive control of each PCR reaction, DNA from the bone marrow of a dog infected with L. infantum culture and water, respectively) were included in each batch.DNA was isolated from 15 mg of spleen tissue, employing the NZY Tissue gDNA Isolation kit, according to the tissue protocol instructions, and later eluted in 50 µL of elution buffer. Positive and negative controls  and later visualized under UV light. After each PCR protocol, the samples were considered positive if a band of the expected size was observed in the gel. Each DNA sample was subjected to five protocols of PCR to detect the parasite: Two protocols targeting kinetoplast DNA (kDNA) ,29, one https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 16 April 2021). Sequences of more than 200 bp were submitted to GenBank for accession number identification.Positive samples were cleaned using an ExoSAP-IT commercial kit . Amplicons were sequenced in both directions at the Genomic Unit of Complutense University of Madrid with a 3730 × L DNA analyzer (Applied Biosystems), using a BigDye Terminator Cycle Sequencing kit v3.1 (Applied Biosystems). The obtained sequences were aligned employing Lasergen SeqMan software version 7.0.0  and manually checked. The consensus sequences were compared with available sequences using the BLAST algorithm of NCBI  (PCRs of the Repeat region and kDNA (RV1/RV2 primers) were able to detect 0.5 promastigotes of  pg DNA) , while L−4-fold dilution from the original DNA  and LnPCR SSU appeared less sensitive, amplifying only at a maximum of a 10−2-fold dilution from the original DNA sample. When DNA from the bone marrow of a positive dog was employed, PCR of the Repeat region and of kDNA (RV1/RV2 primers) were again the most sensitive protocols, displaying positive results even at a 10inal DNA . PCR of In total, 22 samples were analyzed by the five PCR protocols. Only PCR of the Repeat region and LnPCR of SSU rendered positive results, while the other three techniques amplified only the positive controls included in each reaction set. Nineteen samples were positive to PCR of the Repeat region, while only two samples were positive to LnPCR SSU .L. infantum sequence L42479. The two 322 bp sequences from the SSU region shared 100% identity with L. infantum sequence MN757921 from an isolate obtained from a dog.The sequences obtained in this study were deposited to GenBank under accession numbers MW972061–MW972074, MW945401, and MW945402. Fourteen 217 bp sequences from the Repeat region were obtained, while four samples displayed shorter sequences and could not be sent to GenBank. The obtained sequences from the Repeat region shared 100% identity with L. infantum, but according to the results obtained employing the different PCR targets, and considering the detection limit obtained, it is probable that the animals carried a low amount of parasites in their bodies. All positive animals, except one, were adults, and they had exposure to at least one full season of sandfly activity. An explanation for the low parasite load could be that the context in which minks interact with sandfly populations in the wild limits the amount of bites they are exposed to and, consequently, the dose of promastigotes that each animal received. Alternatively, a transient infection with the parasite could be the cause of the low parasitic load. American minks have a lifespan of 10–12 years in the wild and they live in colonies, which facilitates host–sandfly contact. These characteristics, together with the high percentage of infection, the presence of Phlebotomus perniciosus in the area [L. infantum DNA sequence from a dog, support their role as a host of the parasite and they should be considered for future studies.Herein, we reported a high percentage of animals infected with the the area , and theL. infantum has been described [L. infantum DNA: European mink (Mustela lutreola), Eurasian otter (Lutra lutra), Beech marten (Martes foina), European pine marten (Martes martes), domesticated ferrets (Mustela putorius furo), European badger (Meles meles), and European polecat (Mustela putorius) [There are a large number of domestic and wild animals in Europe, besides dogs, in which escribed . Among tescribed . In addiutorius) ,36,37,38L. infantum seems to be frequent in farmed animals, only one study detected a wild American mink infected with the parasite by qPCR in Spain, but the authors only tested one animal [A high rate of infection has been reported in farmed American minks , even at a young age . In anote animal . In all L. infantum sequences from humans and animals. Unfortunately, only two of them displayed positive results. Under our conditions, PCR using the Repeat region and kDNA (RV1 and RV2 primers) were the most sensitive techniques for detecting DNA from parasite cultures and from the bone marrow of a positive dog. Using DNA samples from cultures, in which the amount of parasites is easier to quantify, LnPCR of SSU showed the same limit of sensitivity. When employing PCR from the ITS region, a lower percentage of positivity has also been reported by other authors, including lower values of PCR detection in farmed American minks compared to serology [In this study, we employed different targets to detect the parasite as a multi-locus approach to assess the identity of the obtained sequences in comparison to other serology . Lower sserology . In agreserology , PCR ampOther authors have also compared different protocols of amplification, with the results varying between studies, although most of them agreed in pointing out that kDNA is a high-sensitivity target due to its multicopy nature, with thousands of maxicircles and several dozen of minicircles . LachaudAlbuquerque et al.  comparedConcerning the negative results obtained with both kDNA PCR protocols, they might be due to negligible changes in DNA sequences being enough to avoid amplification from the samples, since they represent a more variable target than the Repeat region or the SSU region . Indeed,L. infantum is necessary. Different sensitivities of the protocols, or minimal differences in the DNA sequences of more variable targets, could interfere with the results.Taking into account our results, we believe that the employment of more than one target to detect L. infantum in communities of wild American minks. The American mink is an invasive species that menaces the European mink, but it also harbors several zoonotic infections [This is the first report of the detection of fections ,17,20,21


In the control group, there was a higher rate of side effects and a significantly longer hospitalisation time (p < 0.001). Similarly, higher satisfaction with the applied analgesic treatment was noted in the FICB group. Conclusions: FICB in elective THR treatments is an effective form of analgesia, which reduces the need for opioids, the number of complications, the length of hospitalisation, and which ensures a high level of patient satisfaction with the analgesic treatment used. Trial registration: ClinicalTrials.gov No. NCT04690647.Objective: An assessment of the feasibility of fascia iliaca compartment block (FICB) combined with nonopioid analgesics and patient controlled analgesia (PCA), oxycodone, in the perioperative anaesthetic management for elective total hip replacement (THR). Design: A randomised, single-center, open-label study. Setting: A single hospital. The study was conducted from October 2018 to May 2019. Participants: In total, 109 patients were scheduled for elective total hip replacement. Interventions: Postoperative FICB with 0.375% ropivacaine in conjunction with nonopioid analgesics  and oxycodone as rescue analgesia. Measurements: Pain intensity was measured using the Numeric Pain Rating Scale (NRS) at rest and during rehabilitation, the total dose of postoperative oxycodone required, the occurrence of opioid-related adverse events, patient hospitalisation time, and level of satisfaction. Follow-up period: 48 h. Main Results: A total of 109 patients were randomised into two groups and, of these, 9 were subsequently excluded from the analysis . Patients in the FICB group received standard intravenous analgesia with FICB, and those in the control group were managed with standard intravenous analgesia only. Pain level measured with NRS was significantly lower at rest and during rehabilitation in the FICB group. Oxycodone use in the first 48 h was significantly higher in the control group ( The development of joint reconstructive surgery is considered to be one of the most important advances in interventional orthopaedics in recent years ,2. TotalOne of the fundamental elements of modern hip reconstructive surgery is to ensure optimal analgesia while minimising the need for opioids and reducing adverse effects associated with their systemic administration. The most common adverse reactions include postoperative nausea and vomiting, oversedation, apnoea, and respiratory complications. Early rehabilitation and efficient mobilisation of the patient is another element of perioperative care in joint replacement surgeries ,8.Regional anaesthetic techniques and local infiltration of the surgical site using local anaesthetics (LAs) are the primary elements of modern, multimodal treatment approach for acute postoperative pain, contributing to both improved control of postoperative pain and early patient mobilisation and rehabilitation ,10,11,12Fascia iliaca compartment block (FICB) is one of the regional nerve blocks used in THR ,15,16. IThe main aim of the study was the assessment of FICB efficacy in elective THR. The following parameters were assessed: NRS at rest and during rehabilitation, the need for opioids, adverse effects related to their systemic administration, time to first analgesic intervention, and patient satisfaction with the analgesic regimen.2, no contraindications for postoperative pharmacotherapy, and analgesia used in the study. Patients with contraindications for spinal anaesthesia and regional blocks, previously diagnosed with chronic pain, chronic use of opioids, obesity (BMI > 30 kg/m2), allergy to drugs used in the study, and mental state preventing proper use of patient-controlled analgesia (PCA) pump were excluded from the study.This was a prospective, observational, randomised study. The study was approved by the Ethics Committee of the District Medical Chamber in Kielce, Poland  and was registered under Clinical Trial No. NCT04690647. Patients qualified for elective posterolateral hip replacement and hospitalised in the Saint Lucas Hospital in Końskie between October 2018 and May 2019 were included in the study. The inclusion criteria were as follows: age > 18 and < 75 years, American Society of Anaesthesiology (ASA) physical status I–III, BMI 19–30 kg/mControl group—standard intravenous postoperative analgesia,FICB group—standard intravenous postoperative analgesia + ultrasound-guided supra-inguinal fascia iliaca compartment block (S-FICB).Patients meeting the inclusion criteria were randomised to one of two groups receiving different postoperative analgesic regimens:www.random.org website.Having been thoroughly informed about the aims and method of the study, and after giving informed consent, the patients were randomly allocated to the control group or the FICB group. The allocation numbers were computer-generated using the The pre- and intra-operative management protocol was standardised for all patients qualified for the study. No standard premedication was used. All patients received preventive analgesia in the form of oral paracetamol  at 500 mg, oral metamizole  at 500 mg, and oral pregabalin  at 75 mg an hour before the procedure. After arriving at the operating room and starting standard monitoring  within the block room, spinal anaesthesia was performed. For this purpose, the patient was placed in a sitting knee-chest position. Then, after surgical disinfection of the puncture site (L2/L3 or L3/L4), the subarachnoid space was identified at midline using a needle for spinal anaesthesia  under sterile conditions. After clear, colourless cerebrospinal fluid appeared, 1.7–2.2 mL of 0.5% hyperbaric bupivacaine solution  was injected. The extent of the sensory block was assessed using an aerosol disinfectant. Once the optimal extent of sensory blockade (up to the level of Th10) was achieved, the patient was transported to the operating room and placed in a lateral position to perform the procedure.During the surgery, sedation using IV propofol  at 25–50 µg/kg/min or headphones connected to an audio system (relaxing music) were used on patient’s request ,19. StanAfter the procedure, the patients stayed in the postoperative supervision room and were monitored for vital parameters. After obtaining a minimum score of 9 on the Aldrete’s Scoring System twice, approximately 30 min apart, the patients were discharged to the surgical unit. All patients in the postoperative supervision room additionally received oxycodone  in the PCA system with a bolus of 1 mg and a 10-min lock-out. The patients also received an information brochure on the use of the PCA pump and were instructed on how to use the pump. Standard multimodal analgesia with the use of nonopioid and opioid analgesics was used in the study groups  compared to FICB patients (6.6 ± 0.7 vs. 5.6 ± 0.9) .We found differences in patients’ satisfaction with the analgesic treatment used between the groups. A total of 80% of FICB patients were very satisfied or satisfied with the postoperative analgesia compared to only 30% in the control group. No patients were reporting to be ‘very dissatisfied’ with analgesia in the FICB group compared to 6% in the control group.Although almost a decade has passed since the first description of FICB , reportsIn the case of patients qualified for elective THR, FICB was found to significantly reduce the need for postoperative opioids ,26. DesmAlthough the use of opioids has been an unquestionable state of the art in anesthesia and postoperative pain management, there is emerging evidence that it can be associated with many sides effects not only in the immediate intra- and postoperative period , but also affects long-term outcomes and influences the entire life of patients, like potentially developing opioid dependence or opioid-induced hyperalgesia ,28. TherPostoperative nausea and vomiting, reduced gastrointestinal motility, and excessive somnolence are the main postoperative adverse effects related to systemic opioids. These adverse effects were significantly more common in the control group due to the increased use of oxycodone. Similar to our study, Xiao-yan Zhang and Gao Yun showed a similar difference in the incidence of opioid-induced adverse effects in their meta-analyses, which included 372 and 325 patients undergoing THR, respectively ,29.Discharge after hip replacement depends on the fulfillment of several key criteria. It is recommended that patients may be discharged if they are mentally and physically able to continue rehabilitation, move about, and travel over short distances . Our anaAmong the many factors affecting patient’s satisfaction with the treatment process, the degree of control of nociceptive experiences in the perioperative period is a key factor ,32. An aThere are some limitations to our study. First of all, the sample size was small and the results should be confirmed in a large study. Also, further studies should be designed as multi-centre studies to exclude local factors affecting the final results. Another limitation is restricting the time of follow-up to 48 h without taking into account the long-term observation. Extending the follow-up to 1, 3, and 6 months would allow for observing the development of chronic pain in individual groups. Furthermore, the use of various adjuvants, which prolong the action of the local anesthetics used, could improve the results obtained. Open label design of the study was also a significant study limitation. Patients and assessor blinded design of the study would probably be more effective in reducing the risk of potential responder bias. Another imitation was the lack of a uniform patient sedation protocol (propofol infusion or headphones with relaxing music) that would translate into change in postoperative pain.Compared to intravenous multimodal analgesia, supra-inguinal fascia iliaca compartment block (S-FICB) is an effective form of analgesia for elective posterolateral total hip replacement. It reduces the need for opioids, and thus the rates of complications resulting from their systemic administration. It also reduces the length of hospital stay and ensures a high level of patient satisfaction with the analgesic treatment used. The obtained results allow for recommending S-FICB for an elective posterolateral hip replacement as an effective and safe method of postoperative analgesia.


Implementation of reliable methodologies allowing Reduction, Refinement, and Replacement (3Rs) of animal testing is a process that takes several decades and is still not complete. Reliable methods are essential for regulatory hazard assessment of chemicals where differences in test protocol can influence the test outcomes and thus affect the confidence in the predictive value of the organisms used as an alternative for mammals. Although test guidelines are common for mammalian studies, they are scarce for non-vertebrate organisms that would allow for the 3Rs of animal testing.Caenorhabditis elegans. Small organisms like C. elegans are upcoming in new approach methodologies for hazard assessment; thus, reliable and robust test protocols are urgently needed. A literature assessment of the fulfilment of the reporting criteria demonstrates that although studies describe methodological details, essential information such as compound purity and lot/batch number or type of container is often not reported. The formulated set of reporting criteria for C. elegans testing can be used by (i) researchers to describe essential experimental details (ii) data scientists that aggregate information to assess data quality and include data in aggregated databases (iii) regulators to assess study data for inclusion in regulatory hazard assessment of chemicals.Here, we present a set of 30 reporting criteria as the basis for such a guideline for Developmental and Reproductive Toxicology (DART) testing in the nematode  Developmental and Reproductive Toxicity (DART) is a critical safety evaluation of chemicals under chemical legislations throughout the world. In the European context, chemicals are controlled under REACH . The goal of REACH is to protect human health and the environment. In the EU, a chemical compound cannot be used or imported until sufficient information has been provided regarding the safety of the compound . In casein vitro tests, e.g. skin and eye irritation tests. For evaluating DART, the OECD guidelines describe pre-, post-, and perinatal development, and multiple generation testing in rodents and non-rodent mammals . These guidelines include instructions on how to carry out safety testing in rodent and non-rodent animal models, as well as in  mammals . For exaThere are drivers to Replace, Reduce, and Refine testing with vertebrate animal models (3Rs), the come from the public, governmental authorities, the scientific community, and industry. Besides ethical reasons, data from animal tests may have limited translational value for humans health , 5. OtheCaenorhabditis elegans, the zebrafish embryo Danio rerio, and the slime mold Dictyostelium discoideum. However, no standardized protocols currently exist describing how DART tests should be carried out in NAMs and which quality criteria should be fulfilled to obtain a reliable result. Reporting guidelines describing what experimental information should be reported for DART in order to independently judge the quality of a study are also lacking.New Approach Methodologies (NAMs) can provide a first decisive tier in hazard assessment through DART screening of compounds, after which the safety of only a limited set of compounds is further investigated by vertebrate tests . PromisiC. elegans. Beronius et al. [in vivo toxicity studies, including DART. However, these criteria cannot be readily applied to tests using C. elegans, since these were formulated with vertebrates in mind. For example, C. elegans nematodes are not easily identified individually, and bedding and water bottle conditions do not apply to C. elegans tests. Guidelines that do take NAM species into account are the ARRIVE 2.0 guideline , updated by the UK National Centre for the 3Rs (NC3Rs). These guidelines describe publication considerations for animal research, including mammalian species and model organisms such as Drosophila melanogaster and C. elegans [. Specific C. elegans guidelines for reliable results in toxicity testing by good C. elegans culture practice (GCeCP) are described by Hunt [Guidelines related to NAM species that are not specific to DART are OECD 212 and 236, describing short-term toxicity and acute toxicity in fish embryos to assess aquatic toxicity. In addition, ISO 10872 describes tests for toxic effects of sediment and soil samples on growth, fertility, and reproduction of  elegans . SpecifiC. elegans, and (iii) assess the reporting quality of DART studies based on those guidelines.This in-depth review aims to (i) get insight into the representation of NAM species in recent publications of DART tests (ii) formulate a reporting guideline to assess DART specifically in C. elegans DART test guideline.The quality criteria were formulated starting from the SciRAP criteria developed by Beronius et al. (2014). We then performed a comprehensive literature search, screened abstracts for inclusion, and assessed the reporting quality of eligible publications according to the pre-set criteria. Most studies fulfilled the majority of criteria, and some criteria are fulfilled by all studies. We conclude that there is substantial consensus in the field on what methodological details are important to report, which is an essential first step towards developing a future C. elegans, and D. discoideum. We limited our search to records published after January 2018 to obtain the most current practice. We analysed the C. elegans studies in-depth, by manually pre-screening records related to C. elegans based on the title and abstract and, if deemed relevant, further screening based on the full text. The criteria to exclude the study based on the abstract were (i) no DART outcomes reported (ii) no chemical exposure data, (iii) not species of interest, and (iv) not a primary study.The complete search strategy is presented in The full text of the study was included in the review if it met the following inclusion criteria: (i) DART outcomes reported (ii) chemical exposure data (iii) species of interest and (iv) wild-type animals.Two readers each read half of the full-text articles.C. elegans experiments optimized for guideline acceptance and data processing. The team includes (i) experts in in the field of C. elegans research , (ii) experts in the field of OECD guideline studies and risk assessment in the field of registration studies for pharmaceuticals and chemicals (MR), OECD guideline studies for regulatory in vitro (geno)toxicity testing for the registration of chemicals, food ingredients, and pharmaceutical products (CK), and formal validation and pre-validation studies for inclusion of tests in the OECD, related to ECVAM activities (MT), and (iii) experts in the field of data science and bioinformatics , with experience in meta-research, including comprehensive searching. The TIER-I and -II criteria from SciRAP were the basis for formulating the C. elegans criteria. These criteria were adjusted and amended to fit the needs of studies in C. elegans.A team of interdisciplinary experts was assembled to formulate a reporting guideline for All full-text articles were scored for the presence of details on each of the 30 reliability criteria . The detD. discoideum, and 191 publications for C. elegans. This suggests that most of the recent literature is focused on established model organisms such as rats and still relatively few studies are based on non-mammalian model organisms  publications to assess reporting quality based on the full text (C. elegans (http://www.scirap.org/ downloaded 24 May 2017) [C. elegans studies  , becauseay 2017) . We adapWe formulated new criteria that are critical in evaluating the quality of the assay that is being performed: the composition of the media during exposure; there are various ways of culturing nematodes; this can be performed in solid agar or liquid medium. The uptake of the compound (and thus the exposure) depends on many variables and can vary based on the media used . For exaadministration method, e.g., if oral: via feed, gavage, drink from pipette, etc’ to ‘administration method, e.g., passive dosing rings/discs.’ Some criteria were unchanged by do have special applications in C. elegans, for example food type and source. The food source is usually E. coli bacteria or axenic medium for nematodes; it should be described whether the bacteria are alive or dead and how much food was provided. This can influence study outcomes through the metabolization of compounds during exposure by bacteria [Some criteria were rephrased, such as: ‘bacteria , live cubacteria , killingbacteria , the bacbacteria , and bacbacteria . It is iC. elegans DART publications whether they reported each of the criteria in the different categories , duration of administration (96% 46/48), the age and life stage of animals at the start of administration such as L1 or L4 (92% 44/48).We scored the 48 tegories , Fig. 2.E. coli bacteria are often fed to the nematodes, and living bacteria can take up the test compound and metabolize it [et al. [For culturing conditions, most studies missed a lot of information. Generally, temperature (92% 44/48), composition of the media (92% 44/48) and food type are reported (90% 43/48). However, information on the amount of food (19% 9/48) as well as information on the state of the food  was often missing . Describolize it , therebyolize it . The rouolize it . For tol [et al. , may hav [et al. . For cor [et al. . The matThe details of examinations are described in all studies (100% 48/48). However, reproductive toxicity is assessed by different parameters, such as number of eggs, number of hatching eggs, the sum of eggs and larvae, hatching time, number of nematodes surviving to each developmental stage—also, the time when the number of offspring was counted greatly differed between studies. Further standardization may be needed for reproducible DART test results. In addition, it is vital that details of adverse events in both dosed populations and controls, including number of Dauer stage nematodes are reported, as their presence can reduce nematode growth. In addition, population composition in terms of life stages and sex, and the method of measuring these parameters should be reported.The most complete publications, reported on 26 out of 30 criteria , 32 Fig.et al. [A good example of quality reporting is Brunquell et al. , a studyet al.  can be fet al. . At thiset al. .C. elegans reporting criteria in We consider the implementation and reporting of quality criteria and well-defined test methods a critical factor for reliable implementation of NAM test methodologies as alternatives for mammalian testing not only for DART but also for testing other types of toxicity or even health-promoting effects. We provide a framework for relevant and essential C. elegans is currently limited. Nevertheless, C. elegans studies could provide valuable information on DART, given that studies are carried out according to quality criteria. Here we propose a set of 30 criteria, for which details need to be described in a study about DART in C. elegans. We assessed existing literature for current adherence to these criteria. We found that researchers almost always include information about the compounds, such as the manufacturer, although specific details such as batch numbers and purity are mostly missing. The culture conditions are often not described in enough detail to judge if the experimental outcome is reliable and reproducible. Proper testing is essential for the success of NAM to be embraced in the future to ensure reliability. With the provided framework of essential reporting criteria, we aim to advance the reduction, refinement, and replacement of animal testing using C. elegans in a reliable manner in the future.Literature of the last 2 years shows that the number of studies on DART in Guideline_Supplementary_information_tfab109Click here for additional data file.