We aimed to establish and validate a nomogram for predicting the disease-specific survival of invasive lobular carcinoma (ILC) patients.The Surveillance, Epidemiology, and End Results program database was used to identify ILC from 2010 to 2015, in which the data was extracted from 18 registries in the US. Multivariate Cox regression analysis was performed to identify independent prognostic factors and a nomogram was constructed to predict the 3-year and 5-year survival rates of ILC patients based on Cox regression. Predictive values were compared between the new model and the American Joint Committee on Cancer staging system using the concordance index, calibration plots, integrated discrimination improvement, net reclassification improvement, and decision-curve analyses.In total, 4155 patients were identified. After multivariate Cox regression analysis, nomogram was established based on a new model containing the predictive variables of age, the primary tumor site, histology grade, American Joint Committee on Cancer TNM (tumor node metastasis) stages II, III, and IV, breast cancer subtype, therapy modality (surgery and chemotherapy). The concordance index for the training and validation cohorts were higher for the new model  than for the old model (0.733 and 0.779). The new model had good performance in the calibration plots. Net reclassification improvement and integrated discrimination improvement were also improved. Finally, decision-curve analyses demonstrated that the nomogram was clinically useful.We have developed a reliable nomogram for determining the prognosis and treatment outcomes of ILC. The new model facilitates the choosing of superior medical examinations and the optimizing of therapeutic regimens with cooperation among oncologists. ILC has unique clinical, pathological, and radiographic features that suggest it is a distinct clinical entity. Over the last 2 decades ILC has accounted for 25,000 to 30,000 new cases of breast cancer in the USA annually, and its incidence is increasing, especially among postmenopausal women. If considered an independent cancer type, ILC would be the sixth-most-common cancer in women, with an occurrence frequency similar to those of non-Hodgkin's lymphoma and melanoma.,5 ILC tumors typically have a good prognosis, low histology grade, and positivity for the estrogen receptor; however, they can be strongly metastatic and are the main cause of cancer deaths among women in many countries worldwide.,6 There is increasing evidence that ILC is clinically unique, and that its early diagnosis and prognosis are especially important.Breast cancer is a heterogeneous disease with multiple prognoses.The American Joint Committee on Cancer (AJCC) staging system has been widely used to determine clinical treatment strategies and assess clinical risks. However, there are limitations in using the AJCC staging system alone to predict the prognosis of patients, and the overall outcomes can vary widely for tumors at the same stage. The clinical uniqueness of ILC means that novel prognostic tools are needed to increase the accuracy of predicting the survival of affected patients. A nomogram can therefore be an effective visual tool for improving the predictive accuracy of the prognosis in individual patients and for providing individualized prognostic information based on a combination of parameters.–11 Nomograms have been found to be helpful for clinicians making decisions and predicting the outcome of an individual, thereby bringing benefits to both clinicians and patients. The aim of this study was to establish a comprehensive prognostic evaluation system for ILC patients and validate its predictive accuracy.A nomogram is a convenient diagrammatic representation of a mathematical model that combines various important factors to predict a specific endpoint.22.1Patient information was collected from the Surveillance, Epidemiology, and End Results (SEER) database, which covers approximately 30% of the population of the USA and includes cases from 18 registries. Informed patient consent is unnecessary when utilizing data from the SEER program that does not include personal identifying information. We searched for ILC patients using the ICD-O-3  histological subtype code 8520/3. We used the sixth edition of the AJCC staging system and restricted our search to between 2010 and 2015.2.2The analyzed demographic variables of the patients included age at diagnosis, race, sex, marital status, primary tumor site, histology grade, laterality, AJCC tumor node metastasis (TNM) stage, AJCC T stage, AJCC N stage, AJCC M stage, treatment status , bone metastasis, and breast cancer subtype.380 years. Race was classified into white, black, and other. Sex was classified into female and male. Marital status was classified into married, unmarried, and unknown. The primary tumor site was classified into the axillary tail, central portion, lower-inner quadrant, lower-outer quadrant, upper-inner quadrant, and upper-outer quadrant of the breast. The histology grade was classified into grades I, II, and III. Laterality was classified into left primary origin, right primary origin, and only 1 side. The AJCC TNM stage was classified into stages II, III and IV. The AJCC T stage was classified into stages T1, T2, T3, and T4. The AJCC N stage was classified into stages N1, N2, and N3. The AJCC M stage was classified into stages M0 and M1. Surgery, radiation, and chemotherapy were classified into receiving and not receiving/unknown. Bone metastasis was classified into yes and no/unknown. The breast cancer subtype was classified into luminal A, luminal B, HER2 enriched, and triple negative. Patients with missing or unknown survival time were excluded.Age was classified into <40, 40 to 59, 60 to 79, and 2.3Continuous variables that conformed to a normal distribution were expressed as mean ± SD values, while categorical variables were expressed as frequencies and percentages. Multivariate Cox proportional-hazards regression models were applied to determine the factors associated with survival. Based on the predictive model with the identified prognostic factors, a nomogram was constructed for predicting the 3-year and 5-year survival rates of ILC patients.P < .05 considered to be indicative of statistical significance.The nomogram was tested by measuring discrimination and calibration curves in both a training cohort  and a validation cohort . The predictive accuracy of the nomogram was evaluated using the concordance index (C-index) and the area under the time-dependent receiver operating characteristic curve (AUC). The C-index quantified the predictive ability of the model, and ranged from 0.5 to 1.0. Calibration plotting was used to evaluate the agreement between the predicted probabilities and the actual outcomes. Bootstrapping with 500 resamples was used to evaluate both discrimination and calibration. The relative integrated discrimination improvement (IDI) and the net reclassification improvement (NRI) were calculated to estimate the accuracy of the model in predicting outcomes with and without the application of prognostic therapies. Decision-curve analyses (DCAs) were used to assess the clinical value of the predictive models. All statistical analyses were performed using SPSS  and R software (version 3.6.1), with a 2-sided probability value of 2.4Because of cancer is a reportable disease in every state of the USA, informed patient consent is not required. Once the data use agreement is signed, data on cancer research is freely available to the public.33.1There were 4155 eligible ILC patients identified in the SEER database. For nomogram construction and validation, we randomly assigned 70% of the patients to the training cohort (n = 2908) and 30% to the validation cohort (n = 1247). The survival period was known for all of the patients included in this study. The largest proportions of the patients were aged 60 to 79 years , white (83.5% and 83.1%), female (both 99.9%), married (82.6% and 80.0%), and had a primary tumor site in the upper-outer quadrant of the breast (57.2% and 57.3%). Many patients were histology grade II, AJCC TNM stage II, AJCC stage T2, AJCC stage N1, AJCC stage M0, and luminal-A breast cancer subtype. About 94% of patients had undergone surgery. More than half of the patients had received both radiation and chemotherapy. Table 3.2P < .001), AJCC TNM stage IV , no surgery , no chemotherapy , and triple-negative breast cancer subtype were independent risk factors for the survival of ILC patients  on this nomogram was then converted into the probabilities of 3-year and 5-year survival. The nomogram showed that the AJCC stage was the most important contributor to the prognosis, followed by the breast cancer subtype, surgery status, primary tumor site, age, histology grade, and chemotherapy status Fig. .3.4The C-indexes were higher for the nomogram  than for the AJCC staging system (0.733 and 0.779). For the nomogram, the AUCs of the training cohort (0.793 at 3 years and 0.772 at 5 years) and validation cohort  indicated that the model had better discriminative ability than the 6 edition of the AJCC staging system  and 0.117 (95% CI = 0.074–0.278) for 3 years and 5 years of follow-up examinations, respectively, in the training cohort; the corresponding values in the validation cohort were 0.269 (95% CI = 0.110 to 0.446) and 0.180 (95% CI = –0.024 to 0.345). These values indicate that the nomogram represented a great improvement. Similarly, the IDI values for 3 years and 5 years of follow-up were 0.028 and 0.030 in the training cohort, respectively, and 0.040 and 0.033 in the validation cohort, which indicate that the new model had superior predictive performance.3.6The results of the DCA graphically showed that the new model yielded greater net benefits for the 3-year and 5-year survival than the traditional AJCC staging system, which indicates that the model is clinical useful and can play a useful practical role in decision-making.4 Although ILC is less common than invasive ductal breast cancer (IDC), the proportion of cases with ILC is gradually increasing.,15 ILC tumors have a better long-term outlook and tend to be less aggressive than IDC tumors, but they are inclined to metastasize to the genital tracts, the gastrointestinal system, and meninges, and additional more commonly transfer to atypical sites.–20 This situation indicates the need for further research into the prognosis of ILC. The early identification of high-risk ILC patients is helpful for providing adjuvant treatment or trials. Although the existing clinical AJCC staging system provides meaningful predictions of the prognosis of ILC patients, it has limitations in estimating the clinical risk of ILC. We have therefore developed a comprehensive predictive model that includes not only the patient demographics but also therapies and other clinical parameters. Our novel model can provide an independent data set for ensuring fairer model assessments.ILC, which is also known as infiltrating lobular carcinoma, is the second-most-common histological type of breast cancer, and its incidence is increasing.–23 In order to obtain reliable research results, we identified 4155 patients with ILC from 2010 to 2015 in the SEER database.This study was based on the large-sample database of the SEER program, which started with 8 registries in 1973 and has continuously increased with the addition of other participating sites over time. Currently the database includes 18 geographically diverse areas representing 30% of the USA population, with efforts made to accurately reflect the racial, economic, and social diversity of the country.–27 The results of the multivariate Cox regression presented in Table ,29 Although ILC often responds poorly or not at all to chemotherapy, there is currently insufficient evidence to support these assumptions.,17,30 Moreover, ILC patients exhibit better disease-free survival and overall survival, especially among those at a high risk.,31 Similarly, from our nomogram  program registries for creating the SEER database.Conceptualization: Rong Fu, Jun Lyu.Data Curation: Jin Yang, ShengPeng Wang, Jun Lyu.Methodology: Hui Wang, Yuzhi Kang, Li Lin, ShengPeng Wang, Jun Lyu.Software: Jin Yang, Rahel Elishilia Kaaya, Jun Lyu.Writing – original draft: Rong Fu.Writing – review & editing: ShengPeng Wang, Jun Lyu.


Measures included substance use, dependence, subjective well-being, social functioning, and mental and physical health. Generalized Linear Models were employed to assess change over time. Results: At 9 months, 68% of participants reported complete 90-day drug abstinence. Alcohol frequency and quantity were reduced by over 50% at 9 months, with 32% of the sample recording 90-day abstinence at 9 months. Both alcohol and drug dependence scores were reduced by over 60%, and small to medium effect sizes were found for a range of psychosocial outcomes at 9 months follow-up, including a doubling of wellbeing scores, and a halving of psychiatric severity scores. Residents who remained in the TC for at least 9 months reported substantially better outcomes. Conclusions: With notably high study follow-up rates (over 90% at 9 months post-exit), these data demonstrate the value of the TC model in achieving substantial and sustained improvements in substance use and psychosocial outcomes for a cohort with severe substance dependence and complex presentations. Implications for optimal length of stay are discussed.The Therapeutic Community (TC) model is considered an effective treatment for substance dependence, particularly for individuals with complex presentations. While a popular approach for this cohort across a number of countries, few studies have focussed on biopsychosocial and longer-term outcomes for this treatment modality. This study reports on substance use, dependence, and biopsychosocial outcomes up to 9 months post-exit from two TC sites. Methods: A longitudinal cohort study ( Originally designed to be implemented in a psychiatric setting, the concept of the Therapeutic Community (TC) has evolved to be applied to drug rehabilitation . CentralTC effectiveness is typically assessed by rates of abstinence from drugs or alcohol, or various measures that indicate a substantial reduction  in use and related harm. However, there is an increasing focus on reporting psychosocial outcomes, consistent with TC philosophy ,8,9, altLength of stay (LOS) in TCs has been linked to improvements in substance use, criminality, employment, and other psychosocial outcomes ,15,16,17Surprisingly, although TCs have been widely implemented across a range of countries, there is a dearth of outcome studies that report post-exit follow-up data greater than six months. One recent multi-site Australian study found that residential rehabilitation (including TCs) was associated with higher odds of abstinence at 12 months follow-up than acute withdrawal services for drugs and alcohol, and higher odds of abstinence than acute withdrawal and outpatient services when alcohol was the primary drug of concern . HoweverThe current study goes some way towards addressing the current gap in the literature by reporting on: TC outcomes specifically , substance use and biopsychosocial outcomes, magnitude and clinical significance of outcomes, and mid- to long-term follow-up data. Specifically, this paper reports on the 3 months and 9 months TC outcome data from a twin-site study conducted in Australia. The primary outcomes examined were: drug and alcohol use; and drug and alcohol dependence. Secondary, biopsychosocial outcomes were: mental health; subjective wellbeing; physical health; social functioning; and employment.Residents who were undergoing residential treatment for substance dependence within a TC facility  were invEthics approval was obtained from the relevant university committee (DUHREC_EC20047) and written informed consent was received from all participants. All participants completed a self-report survey and participated in a face-to-face interview 3–4 weeks post-TC admission . Baseline drug/alcohol use was reported for the 3-month period immediately prior to entering the TC (excluding any incarceration/detoxification period). Interviews were conducted by one of four researchers with post-graduate qualifications in psychology.There were two follow-up assessments: 3 months and 9 months post-exiting the TC. Follow-up interviews were conducted face-to-face; however, where the person was unwilling/unable to attend, interviews were conducted over the phone. This study draws on the data from a funded project examining the impact of a brief alcohol intervention, the findings of which are reported elsewhere .Therapeutic communities are a sub-set of drug-free, residential drug treatment programs. They are highly supervised and structured environments which combine medical, psychological and peer support, with meaningful work activities and therapeutic groups. Initially, residents have minimal contact with external networks as they develop peer relationships and life skills, a positive daily routine and work ethic, and an understanding of their own individual needs. Utilising ‘the community’ as the method or agent of change, TCs comprise a number of stages that individuals work through, leading to increasing self-responsibility, privileges, and responsibility to others. Compared to other types of residential rehabilitation programs, residents in a TC are given greater responsibility for the management and operation of the community, and for providing mutual support and feedback. They contribute to all aspects of the functioning of the community including cooking, cleaning, support, teaching, and maintenance. Consistent with the social identity model of recovery , the undDemographic data were collected at baseline including age, gender, ethnicity/nationality, country of birth, marital status, level of education achieved, occupation, employment status, welfare benefits, living conditions, primary substance of abuse, age at onset of substance abuse problems.Timeline Follow-Back (TLFB): The TLFB method  was usedSeverity of Dependence Scale (SDS): The SDS  has fiveSeverity of Alcohol Dependence Questionnaire (SADQ): The SADQ  is a 20-Opiate Treatment Index (OTI)—social functioning subscale: The OTI  is a selPersonal Wellbeing Index (PWI): The PWI  is an 8-Addiction Severity Index (ASI): The ASI  is a strd effect sizes were calculated for 9 months post-TC exit.Data were analysed using R   and SPSSn = 157) and 90% (n = 150) provided 3- and 9-month data respectively. The majority of 3 months and 9 months interviews were conducted in person . Main reasons for telephone interviews were: no fixed address, incarcerated or living outside metropolitan area.Of the 166 eligible residents who provided baseline data over a 23-month period (2007–2009), 95% (n = 30), the mean number of drinking days was 5.5 per week, with a mean of 25 Standard Drinks consumed per drinking day. The most frequently consumed substances in the 90 days prior to TC entry were alcohol (mean proportion of days = 0.51), cannabis (mean proportion of days = 0.35), heroin (mean proportion of days = 0.24), and amphetamine (mean proportion of days = 0.21). Level of alcohol and other drug dependence was very high in this cohort; for example, the group mean SDS score was higher than scores typically reported in drug treatment-seeking individuals, with 75.3% of all participants scoring 5 and above at treatment entry . For those individuals with a primary alcohol problem, 77% of this subsample (n = 30) were considered severely alcohol dependent (scores above 30 on the SADQ). Baseline wellbeing in this cohort was markedly low, with only 7.2% of residents scoring within the range of normative Australian individual scores , and wi 59,536; ,47,48) (n = 142), 58% were completely abstinent at 3 months and 68% at 9 months, also a large effect size. Further, drug consumption was reduced to over half of baseline levels at 3 months and 9 months in 77% and 82% of the sample, respectively. For the subsample who were not abstinent at follow-up, mean drug episodes dropped by 62% at 3 months and by 52% at 9 months. No significant differences were found for abstinence rates or 50% reduction rates between 3 months and 9 months, indicating that sustained change was achieved by participants.Using GLM, we assessed change in alcohol and drug consumption, and level of dependence at both 3 months and 9 months post TC exit (see n = 30) who identified as having a primary alcohol issue  alcohol frequency was reduced by 70% from baseline to 3 months and 9 months (5.5 drinking days per week at baseline to 1.7 at 3 months and 1.6 at 9 months), with alcohol quantity reduced by 60% from baseline to 3 months and 9 months (25.2 Standard Drinks per drinking day at baseline to 10.2 at 3 months and 10.6 at 9 months). In those with a primary alcohol problem, abstinence rates were 37% at 3 months and 23% at 9 months. In general, the post-treatment alcohol goal for those dependent on drugs was reduction in consumption, rather than abstinence. In this context, 32% of the total sample were completely abstinent from alcohol for the previous 90 days at both 3 months and 9 months. Overall, alcohol consumption was reduced by at least 50% in 63% of the total sample at 3 months and 64% of the sample at 9 months.In terms of alcohol consumption, the number of drinking days was significantly reduced by 60% from baseline to 3 months, and by 57% from baseline to 9 months . At bothd = −0.83), amphetamines (d = −0.79), heroin (d = −0.63), and alcohol  from baseline to 3 months, and maintained at 63% of baseline at 9 months. The percentage of participants scoring above the more conservative clinical cut-off of 5 on the SDS was 73% at baseline, dropping to 36% at 3 months, and 29% at 9 months. For the SADQ, 29% of the sample scored above the cut-off of 31 for severe alcohol dependence at baseline, dropping to 3% at 3 months, and 2% at 9 months. In terms of the sub-sample with a primary alcohol or both a primary alcohol and drug issue, the percentage above this cut-off was 77% at baseline, dropping to 3% at both 3 months and 9 months. Finally, separating consumption episodes by drug type, medium to large reductions across the sample were recorded at 9 months for cannabis , 90 to 179 days , 180 to 269 days , and 270 days or more . Using a negative binomial GLM and correcting for multiple comparisons using the False Discovery Rate method, significant LOS group differences were found for number of drug episodes at 3 months and 9 months. Participants who remained at the TC for longer than 9 months showed a 74% greater reduction in mean drug episodes at 3 months than the mean of the other treatment duration groups combined. At 9 months follow-up, this difference was still significant when comparing participants who remained at the TC for longer than 9 months compared with those who remained for shorter than 3 months, with a 52% greater reduction in drug episodes for the +9 months group compared with the under 3 months group (The median LOS for study participants was 206.5 days (IQR = 104.8–370.0), or approximately 9 months. Participant LOS was grouped as follows: 89 days or less  improved by 112% at 9 months post-exit from the TC. Wellbeing scores at baseline in this cohort (mean PWI = 25.2) were markedly lower than normative scores across 30 general population studies in Australia , and ab 59,536; ,47,48. S 59,536; . However 59,536; . Medical 59,536; , did notA key finding here was that increased LOS was associated with generally better outcomes across a range of domains. This has been shown in a number of previous TC studies, and even while controlling for other predictors ,16,18,19It is critical when examining TC outcome research to understand that people accessing TC treatment tend to have more complex psychosocial concerns than people attending outpatient treatments and other shorter-term residential treatment . The TC Noteworthy strengths of this study are the high rates of baseline recruitment (90% of eligible residents) and follow-up , which are higher than similar studies of complex polydrug users ,54. SubsOne potential limitation was that intake into this study occurred after residents had been at the Therapeutic Community for about 3–4 weeks. Consequently, these data represent a sample who have had a ‘minimum dose’ of at least 3-weeks in the TC. Also, baseline reporting for the period prior to entry to inpatient treatment programs can be affected by both escalated consumption (prior to planned abstinence) or decreased consumption (through preceding detox and other programs). While this may impact data in this study, we addressed this in part by measuring baseline data for the period prior to any detoxification that immediately preceded entry into the TC. Furthermore, due to a lack of control group, it is not possible to separate out the effects of the TC from spontaneous recovery, however, the strong dose–response effect reported in this study indicates that a substantial proportion of the outcomes is likely to be attributable to treatment, rather than to natural recovery. Additionally, previous work has shown that changes in one’s sense of identity (away from a ‘user identity’ and towards a ‘recovery identity’) predict commitment to sobriety and improved wellbeing within TCs . In lineThe findings of this study have some important clinical implications that warrant further investigation. First, despite severe and complex presentations, the majority of TC residents in this study reported marked reductions and/or abstinence in alcohol or drug use, in addition to improvements across a range of psychosocial outcomes. These changes were sustained at 9 months post-TC exit, suggesting significant lifestyle improvements. The large positive outcomes shown in this study reinforce the value of financial investment in providing long-term residential treatment, and the steep linear ‘dose–response’ association suggests that additional funding to support extended length of treatment would come with both economical and personal benefits, given the high relapse rates for this complex population. However, even longer follow-up periods would enable a greater understanding of the degree to which these outcomes are sustained.Second, larger cohort studies could assist in being able to examine moderators and mediators of treatment effectiveness, allowing refinements and improvements to TC program content. Finally, the LOS findings were of interest in the context of the development of ever shorter brief interventions for alcohol and drug treatment. Much work is still needed to understand the optimal treatment length for a particular individual by adopting personalised medicine approaches for outcome studies. However, this study suggests that for treatment-seeking individuals with complex presentations, the widespread three-month treatment benchmark  may be iOverall, the findings show that residential TC treatment of generally severe and complex presentations was associated with substantial and sustained improvements in consumption (across a range of substances), including impressive levels of drug and alcohol abstinence, reductions in dependence, and improvements in several psychosocial factors at 9 months follow up. Of note, these results suggest that extending the length of stay in TC treatment may reap substantial rewards in the long term.


The variables explained 20.1% of the variance in preventive behaviors. The results of this study suggest that better strategies for subjective norms related to parents and issue involvement related to COVID-19 must be considered to improve emerging adults’ preventive behaviors.Emerging adulthood is an important period for establishing health behavior patterns in life. This study aimed to examine factors related to preventive behaviors of emerging adults during the COVID-19 pandemic. A descriptive online survey design was used. Data were collected using a self-administrated, 28-item questionnaire completed by 239 undergraduate students from a university in Seoul, South Korea. The questionnaire was developed based on previous studies and the guidelines of the World Health Organization about COVID-19 preventive behaviors. The mean age of participants was 21.97 years, and the average score for COVID-19 preventive behaviors was 4.13 (SD: ±0.42) on a 5-point scale. Hierarchical regression analyses revealed that subjective norms related to parents (β = 0.425,  The novel coronavirus pandemic (hereinafter COVID-19) is a highly contagious respiratory disease that has spread to more than 120 countries globally after the first infection was confirmed in Wuhan, China, on 1 December 2019. In Korea, the first COVID-19 case was confirmed on 20 January 2020, and as of 20 January 2021, the number of cases has reached almost 80,000 [Apart from vaccines, which are becoming available, there are no specific treatments for COVID-19 to date. Reducing the transmission of the virus is the most effective way to control the pandemic, requiring rapid and widespread behavioral changes that include these three measures: compliance with personal hygiene rules, social distancing, and disease management . CompliaThe “infodemic” is another serious issue related to the increasing use of social media and communication technologies . InfodemTo aid in disease management, innovative solutions such as artificial intelligence (AI), machine learning and deep learning, nanomedicine, novel technologies for vaccine development and therapeutics, novel mathematical modeling, big data, the internet of things (IoT), telemedicine, robots, and 3D printing technology are imperative due to limited resources and the immediate requirements for medical supplies, healthcare support, and treatments . AlthougPsychological responses and subjective norms have been observed to be related to individual protective behaviors in previous global pandemics ,10,11,12Based on recent studies, being older, female, and more educated is associated with a higher chance of adopting the preventive rules . In a stEmerging adulthood has been identified as a new developmental phase, which is characterized by a period of exploration for people 18–29 years old before they settle into established adult roles . During Emerging adults most often experience less severe symptoms but still contribute to the spread of the virus. Within the context of the COVID-19 pandemic, it is crucial to understand the psychological factors of preventive behaviors to manage infections . This stAn online, 28-item questionnaire was developed based on previous studies ,18,28,29The participants were undergraduate students from one university in Seoul, South Korea. The inclusion criteria for the participants were that they be between 18 and 29 years old and that they consented to participate. The necessary sample size was determined to be 194 participants based on G*Power 3.1.9.7 software. G*Power uses Cohen’s effect size measure to determine an appropriate effect size, according to the type of test selected. This study applied multiple linear regression analysis and used the conventional values of Cohen’s effect size for the F-test (multiple regression), which was 0.15. There were 14 independent variables, an effect size of 0.15, an alpha error of 0.05, and a power of 0.95 . We designed an online questionnaire with the Google Docs survey program and uploaded the URL of the questionnaire on the university website. We set the participation limit at 241 (in anticipation of drop-out rates) between 28 and 29 April 2020. The first part of the questionnaire contained instructions and an explanation of the study. It was clearly specified that personal identifying information would not be collected. Ultimately, 239 valid questionnaires were collected. The participation of each student was completely voluntary, and they could withdraw at any stage of the study. Preventive behaviors refer to the preventive actions related to the spread of COVID-19. In this study, preventive behaviors were measured with 10 items addressing hand hygiene (frequency and methods), mobile phone hygiene, wearing masks , coughing etiquette, social distancing, and infodemic behaviors (producing and sharing). Questions were rated on a 5-point Likert-type scale ranging from 1  to 5 . Higher scores reflected good preventive behavior. Perceived susceptibility refers to the perception of being at risk of developing a certain disease . In thisPerceived severity refers to the degree to which an individual is seriously aware of the disease and to what extent it affects their life . In thisIssue involvement was measured with three items addressing importance, interest, and relevance on a 5-point Likert-type scale: “I consider the COVID-19 pandemic as an important issue.”; “The COVID-19 pandemic is an issue that draws attention.”; and “The COVID-19 pandemic is an issue that is highly relatable to me.” The total score ranged from 3 to 15 points, and a higher score indicated higher issue involvement. A previous study showed a Cronbach’s alpha coefficient of 0.88 , while tSubjective norms were measured using 10 items, including 5 items related to normative beliefs and 5 items corresponding to motivations, and rated on a 5-point Likert-type scale. The score was calculated according to the criteria of the item development recommendation of the theory of planned behavior . Higher Data analysis was conducted using the IBM SPSS 25.0 program . Descriptive statistics , an independent t-test, and analysis of variance (ANOVA) tests were used to describe general characteristics, risk perception, issue involvement, subjective norms, and preventive behaviors related to participants’ COVID-19 perceptions. Pearson’s correlation coefficients were calculated for the preliminary examination of the associations between variables. Normal distributions are usually evaluated using plots and means, medians, skewness, and kurtosis. We evaluated skewness and kurtosis to assess the normality assumption.A hierarchical multiple regression analysis was performed with preventive behavior scores as the dependent variable. Hierarchical multiple regression is a form of multiple regression in which the variables are entered into regression based on a predetermined logical scheme. In this analysis, three hierarchical or sequential multivariate regression models were used to examine the factors associated with preventive behaviors related to COVID-19. In the first regression model (Model 1), demographic factors  were entered; following this, the factors of risk perception and issue involvement were entered into the second regression model (Model 2). Risk perception included perceived susceptibility and perceived severity. Further, the factors of subjective norms of friends, school, parents, and official and non-official sources were entered into the final regression model (Model 3). Instructions and an explanation of the study were provided online to the participants prior to completing the questionnaire, and no personal information that would make the participants identifiable was collected. The participants provided written informed consent to participate in the research, and those who completed the survey with valid answers were given a reward of US $2.50. This study was approved (202004-HR-1846-02) by the Research Ethics Committee of the authors’ affiliated university.The general characteristics of the 239 study participants are presented in The means, SDs, skewness, and kurtosis of preventive behaviors, risk perception, issue involvement, and subjective norms are shown in p < 0.001), issue involvement , perceived severity , and schools . The correlations among all the study variables are presented in Correlation analysis confirmed a statistically significant relationship between preventive behaviors and all four dimensions of variables. Preventive behaviors related to COVID-19 showed positive, significant correlations with subjective norms: parents  was 1.08–1.78, and the tolerance limits were 0.1 or more and 10 or less. There was no multicollinearity in the correlation between the independent variables.p < 0.001) was shown to be an important factor in preventive behaviors, with an explanatory power of 1.4% . In Model 2, issue involvement  was identified as an important factor, with an explanatory power of 6.4%. In Model 3, subjective norms related to parents , issue involvement , and sex  were identified as important factors. The model fit was significant , and the explanatory power of the entire model was 20.1% , issue involvement, and subjective norms of parents in promoting preventive behaviors of COVID-19. This study also contributes to research on the infodemic, which is rampant during health crises and is difficult to control. It has gained greater importance with the prevalence of ICTs. This study highlights the importance of mobile phone hygiene in relation to the risk of infection, which requires further research in the future.


KIT, and tyrosine kinase inhibitors are actually available as treatment. However, KIT alteration status in canine oral melanoma still lacks characterization. In this study, we investigated the mutational status and the tissue expression of KIT through DNA sequencing and immunohistochemical analysis, respectively. A homogeneous cohort of 14 canine oral melanomas has been collected, and while tissue expression of the protein was detected, no mutations were identifiable, most likely attributing the dysregulation of this oncogene to a more complex pattern of genomic aberration.Malignant melanomas arising from mucosal sites are very aggressive neoplastic entities which affect both humans and dogs. The family of tyrosine kinase receptors has been increasingly studied in humans for this type of neoplasm, especially the gene coding for the proto-oncogene KIT have been shown, while in COMs only its exon 11 has been adequately investigated. In this study, 14 formalin-fixed, paraffin-embedded COMs were selected considering the following inclusion criteria: unequivocal diagnosis, presence of healthy tissue, and a known amplification status of the gene KIT (seven samples affected and seven non-affected by amplification). The DNA was extracted and KIT target exons 13, 17, and 18 were amplified by PCR and sequenced. Immunohistochemistry (IHC) for KIT and Ki67 was performed, and a quantitative index was calculated for each protein. PCR amplification and sequencing was successful in 97.62% of cases, and no single nucleotide polymorphism (SNP) was detected in any of the exons examined, similarly to exon 11 in other studies. The immunolabeling of KIT was positive in 84.6% of the samples with a mean value of 3.1 cells in positive cases, yet there was no correlation with aberration status. Our findings confirm the hypothesis that SNPs are not a frequent event in KIT activation in COMs, with the pathway activation relying mainly on amplification.Canine oral melanoma (COM) is an aggressive neoplasm with a low response to therapies, sharing similarities with human mucosal melanomas. In the latter, significant alterations of the proto-oncogene  KIT, firstly identified as a homolog of the feline sarcoma viral oncogene v-kit [KIT is also normally expressed in the development of melanocytes [KIT mutant melanocytes could acquire elevated migration abilities. Indeed, given its key role, the gene coding for the KIT protein (KIT) received great attention in the field of human oncology, particularly in the study of neoplasms of melanocytic origin. The genomes of both human cutaneous and mucosal melanomas were deeply studied, and human mucosal melanomas (hMMs) showed a higher frequency of aberrations in the KIT gene rather than their cutaneous counterparts [KIT, with CNAs in 26.3% of cases [KIT mutations have been proposed as an adverse prognostic factor [KIT is affected by CNAs in 6.7% of cases and by SNPs in 1.7% of cases [The proto-oncogene ne v-kit , encodesne v-kit ,3. Even ne v-kit , KIT is anocytes  and it ianocytes  with intterparts ,7,8,9. Hof cases ,11,12,13Mutations identified in hMMs mainly affected exon 11 (Ex-11) ,9, whereKIT mutations [In humans, most tumors affected by utations ,14 mainlutations ), promotGiven the increasing interest in pet dogs as a reliable spontaneous animal model for the study of non-UV-induced melanomas, several authors are also investigating canine melanomas arising in sun-protected sites such as canine oral melanoma (COM), the most frequent neoplasia of the oral cavity in dogs ,16,17. SKIT activation in hMMs, such as exons 13 (Ex-13), 17 (Ex-17) and 18 (Ex-18) [However, regarding the SNP profile of COMs, the majority of the studies focused almost entirely on Ex-11, trying to compare the results obtained in human medicine without investigating other exons that play a key role in  (Ex-18) ,27.When considering the mutations reported in the available human literature, Ex-11 is altered in 67% of hMMs ,30,31,32KIT gene, but no decisive results have been achieved, while a screening of Ex-13, Ex-17, and Ex-18 has not been performed [In veterinary medicine, only mutations affecting Ex-11 have been adequately investigated in the erformed ,34.The tissue expression of the KIT protein has also been investigated by immunohistochemistry (IHC) in various neoplasms, both in human and veterinary medicine  has a better effect on patients affected by tumors bearing SNPs in KIT ,13,27,40KIT , some tyKIT ,20,40, cKIT , and sysKIT .KIT point mutations (77%) compared to patients bearing tumors with KIT amplification (18%) [Moreover, the disease control rate in human patients treated with imatinib, one of the most widely used TKIs, is better in patients bearing tumors with on 18%) , particu% , partion (18%) ,27. For KIT point mutations in Ex-11 [KIT point mutations, disease regression in dogs and cats treated with imatinib and affected by tumors without known KIT point mutations is rare [TKIs are also successfully used in veterinary medicine for the treatment of Mast Cell Tumors (MCTs) with in Ex-11 ,44, and in Ex-11 ,46. More is rare .KIT in COMs at both the gene and protein level. Taking advantage of the cohort of COMs assembled for the array Comparative Genomic Hybridization (aCGH) study published in 2019 [KIT and the protein KIT in our cohort of samples. We performed an accurate evaluation of the possible SNPs by sequencing Ex-13, Ex-17, and Ex-18, and by evaluating KIT expression in COMs. Ki67 was also evaluated by IHC due to its higher expression in metastatic hMMs with SNPs in KIT [In this study, we investigated the role of  in 2019 , we deciKIT somatic point mutations and amplification in COMs.Therefore, the aim of this study was to evaluate and correlate the IHC expression of KIT and Ki67 with Formalin-fixed, paraffin-embedded (FFPE) COMs were collected from different archives. For each patient, anamnestic data including size and location of the tumor were collected. Hematoxylin and eosin (H&E)-stained slides from each sample were checked by 3 experienced pathologists to confirm the diagnosis of COM and to assess the presence of healthy tissue near the pathological mass suitable for the nucleic acid extraction. The diagnosis of COM was made by adopting the criteria proposed by Smedley and colleagues , which w®®) using heptane as the deparaffinization agent and following the manufacturer’s instructions. The extracted DNA was then evaluated on a 1% agarose gel by electrophoresis and quantified with the NanoDrop ND-1000 .With the help of a microtome with disposable blades, 2–3 20 µm-thick slides were cut from each FFPE block. Healthy and cancerous tissues were separated using a clean scalpel and put in 2 different tubes. To avoid any possible contamination, all the instruments were cleaned between one sample and the other. From the paraffinized material, DNA was extracted using the All Prep DNA/RNA Extraction Kit , 40 s at 60.5 °C , 50 s at 72 °C (extension), and 5 min of final extension at 72 °C.The positive control was non-fragmented genomic DNA extracted from fresh-frozen canine cutis, while water was used instead of DNA as the negative control.®®). Using the corresponding forward (Ex-17) or reverse (Ex-13 and Ex-18) primer, amplicons were then sequenced with the Sanger method, and their sequences were visualized with the Chromas 2.6.5 software. Only sequences with high-resolution peaks , minimal baseline noise, and no trace of secondary sequence contamination were considered suitable for mutational analysis. For each sample, healthy and pathological sequences were matched and aligned with the Clustalw platform [All amplicons obtained were separated on a 1.8% agarose gel via electrophoresis to assess the success of the PCR reaction, then purified with ExoSAP  and then tested with an anti-KIT rabbit polyclonal antibody diluted 1:300 .KIT expression was evaluated in each sample included in this study. For each FFPE block, a 4 µm-thick slide was cut, mounted on a polarized glass slide . Both antibodies were previously validated in the canine species [For each specimen, the expression of the Ki67 protein was also assessed with an anti-Ki67 mouse monoclonal antibody diluted 1:50 . To avoid the use of a bleaching reaction which could damage the integrity of the antigens, an ultraView universal alkaline phosphatase RED detection kit  was used (DAB chromogen in unbleached specimens is indeed not usable), and hematoxylin was used as a counterstain.As positive controls, a canine MCT and canine cutis were used for KIT and Ki67 staining, respectively. As negative controls, antibody diluent was applied instead of the antibody.2) within the areas with clear positive staining, starting from the mostly positive field. If no positive cells were found in 3 consecutive hpf, the process was repeated in another IHC-positive area. This method was designed similarly to that used for the establishment of the Ki67 index as described by Bergin and colleagues [The slides were visualized at 40× magnification using a D-Sight scanning machine and the D-Sight Viewer software (A. Menarini Diagnostics). The KIT index was evaluated by counting the mean number of positive cells in 5 consecutive high-power fields . Data distribution was visually checked for normality. To verify mean differences among groups, either the Student’s p < 0.05.The Spearman’s rank correlation analysis was applied to discover associations between variables. The level of significance was set at Data related to the aCGH analysis are published and extensively discussed in the paper from Brocca and colleagues . Twenty The mean age of the 14 dogs selected for the study was 11.98, ranging from 10–17 years. Males represented 78.57%  of the cases, while females represented the remaining 21.43% . Male over-representation is in accordance with the literature ,54. The p < 0.0001, data not shown), and the 260/280 and 260/230 ratios showed variable values, but all were considered of sufficient quality for the amplification steps. All primer pairs were firstly tested on canine control tissues at different temperatures with a gradient PCR, and all pairs showed successful and specific amplification of the selected exons. The amplification was successful in almost all samples for both the healthy and the pathologic tissues. A band at the expected length was obtained in 27/28 (96.4%) reactions  for exon 13, 27/28 (96.4%) for exon 17, and 28/28 (100%) for exon 18  or for the Ki67 index (p = 0.38). Similarly, no differences in terms of KIT and Ki67 expression were noted between males and females , or according to the pigmentation level . Finally, no correlation was found between the two indexes .The KIT index varied from 0.6 to 8.6 in positive samples, with a mean of 3.1 positive cells in 5 hpf. Positive staining for the anti-Ki67 antibody C was obtKIT gene in COMs, particularly in exons 13, 17, and 18, and to correlate the mutational profile of these exons with the amplification status of the gene itself, and with the IHC expression of the KIT protein.The aim of this study was to deepen our understanding of the mutational landscape of the This interest derives from the scarcity of currently available similar studies in the canine species, and from the possible use of pet dogs as a reliable model for the study of hMMs.KIT. Interestingly, the pathway enrichment analysis revealed the enhancement of pathways specifically related to cancer proliferation, but also a significant enrichment of those related to imatinib and drug metabolism. These results indicated that further investigation of the KIT alteration status was warranted.In our previous work  that aimKIT gene in COM, which were successfully PCR amplified and Sanger-sequenced. Taking advantage of the cohort of samples collected for the aCGH study [KIT amplification status. In particular, CFA 13 (comprising the KIT gene) was affected by a copy number gain in 7/20 samples of the original cohort (35%), and we considered it valuable to further analyze the DNA of these seven samples and to compare them with another randomly chosen seven samples that were not affected by the same copy number gain in CFA 13.In this work, we describe the first characterization of the mutational profile of exons 13, 17, and 18 of the GH study , we wereWe developed highly-performing primer pairs and set up a reliable protocol for the amplification and sequencing of short genomic sequences extracted from FFPE blocks.KIT status in COMs does not resemble the mutational status reported in hMMs. This is in line with some of the latest Next Generation Sequencing-based veterinary studies [KIT amplification and coexisting SNPs in exons 11, 13, 17 and 18 in the same tumor [Since no SNPs were detected affecting the examined exons, our study suggests that  studies ,24. Althme tumor ,8.In our study, none of the samples affected by a CFA 13 amplification had an SNP present, and no point mutations have been found at all, suggesting one possible molecular difference between COMs and hMMs.KIT point mutations are considered a sporadic event [The absence of point mutations in our cohort is consistent with the results reported in other recent studies of COMs, in which the ic event ,33,34,47ic event ,10.KIT amplification versus a 0% prevalence of KIT point mutations in our cohort of COMs corroborates the increasingly affirmed hypothesis that the main pathogenesis of COMs and hMMs is related predominantly to CNAs rather than SNPs [The detection of a 35% prevalence of han SNPs ,19,20,21Regarding the pathologic DNA from sample 12, we were not able to obtain an amplification reaction for exon 13 and 17, while the amplification of exon 18 was successful . The reaHere, we proposed a reproducible method for scoring KIT IHC positivity in COMs samples. To date, the IHC evaluation of KIT expression has been limited to a semi-quantitative evaluation, expressed as classes corresponding to an approximate percentage of immunoreactive cells on the total tumor area, often with wide ranges defining a single class ,39. In oKIT amplified and non-amplified samples. This could be due to the low number of cases analyzed, or to the fact that KIT gene amplification does not correspond to a higher KIT protein expression.There was no significant difference in KIT protein expression between As reported by Lassam and Bickford , and by These findings support the hypothesis of Alexeev and Yoon , who proIndeed, a study from Newman et al.  found a Regarding the correlation with other tumor markers, Ma and colleagues  highlighUnfortunately, follow-up data were not available for this cohort of samples, making further evaluations impossible.KIT gene do not present with SNPs in our cohort of COMs, suggesting that they are not involved in the pathogenesis or progression of COM. Moreover, the amplified status was not statistically associated in any way to a mutational profile in the KIT gene, nor to the expression of the KIT protein itself or to the Ki67 marker.In this paper, we showed that exons 13, 17, and 18 of the PTK2, STK3, TEC, PDGFRA, VEGFR2, and CD63, which so far have received less attention.Immunohistochemically, we proposed a quantitative, replicable method for the evaluation of KIT expression, through which 84.6% of the samples (11/13) were detected to be positive for KIT. Although the number of cases included in this study did not allow us to draw definitive conclusions, our findings provide new insights for the current knowledge on the use of pet dogs as a spontaneous model for the study of hMMs. Further studies with a greater number of cases are needed to clarify unresolved questions, including the role played by other kinases that have also been found to be altered in our cohort via aCGH analysis, such as


Oxidative stress is thought to play a significant role in the development of glaucoma. However, the association between systemic and local oxidative stresses in different types of glaucoma has not been assessed fully. The current study compared the redox status in the aqueous humor (AH) and blood samples among eyes with primary open-angle glaucoma (POAG), exfoliation glaucoma (EXG), and non-glaucomatous controls to evaluate the relationship among systemic redox status, intraocular oxidative stress, and clinical backgrounds. AH and blood samples were obtained from 45 eyes of 45 Japanese subjects . The serum levels of lipid peroxides, ferric-reducing activity, and thiol antioxidant activity were measured by diacron reactive oxygen metabolites (dROM), biologic antioxidant potential (BAP), and sulfhydryl (SH) tests, respectively, using a free radical analyzer. The activities of cytosolic and mitochondrial forms of the superoxide dismutase (SOD) isoforms, i.e., SOD1 and SOD2, respectively, in AH and serum were measured using a multiplex bead immunoassay. In AH, SOD1 in subjects with EXG and SOD2 in those with POAG and EXG were significantly higher than in control eyes. In serum, compared to control subjects, BAP in subjects with POAG and EXG was significantly lower; SOD1 in those with EXG and SOD2 in those with POAG and EXG were significantly higher. dROM and SH did not differ significantly among the groups. The BAP values were correlated negatively with the SOD1 concentrations in AH and serum, SOD2 in the AH, intraocular pressure, and number of antiglaucoma medications. In conclusion, lower systemic antioxidant capacity accompanies up-regulation of higher local antioxidant enzymes, suggesting increased oxidative stress in eyes with OAG, especially in EXG. Determination of the systemic BAP values may help predict the redox status in AH. Glaucoma, which is a leading cause of irreversible blindness worldwide ,2, is a Oxidative stress is defined as an imbalance between oxidant generation and antioxidant response . The excThe current study compared the redox status in the AH and serum among patients with primary OAG (POAG), exfoliation glaucoma (EXG), and non-glaucomatous controls. We also assessed possible associations between systemic and local redox status and clinical parameters.The current study procedures were carried out in accordance with the Declaration of Helsinki. The institutional review board of Shimane University Hospital approved the research (No. 20091119-1 and No. 20131216-1). All participants provided written informed consent for inclusion in the study. This study included 15 eyes of 15 patients  with POAG (including 1 pseudophakic eye), 15 eyes of 15 patients  with EXG (including 2 pseudophakic eyes), and 15 eyes of 15 patients  with only senile cataracts as controls, who had undergone cataract and/or glaucoma surgery. All surgeries took place at Shimane University Hospital from May 2017 to February 2018. All participants underwent ophthalmologic examinations including measurements of the best-corrected visual acuity (BCVA); IOP measured by Goldmann applanation tonometry; and slit-lamp, gonioscopic, and pupil-dilated funduscopic examinations. Briefly, we diagnosed POAG based on the findings including bilateral open iridocorneal angles and a characteristic appearance of glaucomatous optic neuropathy such as enlargement of the optic disc cup or focal thinning of the neuroretinal rim, corresponding visual field defects detected by the Humphrey Visual Field Analyzer Swedish Interactive Thresholding Algorithm central 30-2 program  in at least one eye, and no evidence of secondary glaucoma bilaterally. We diagnosed EXG in the presence of exfoliation material deposition on the anterior lens capsule and/or at the pupillary border observed by slit-lamp examination, IOP > 21 mmHg, and/or glaucomatous optic neuropathy with consistent visual field changes. The controls had senile cataract, no glaucomatous optic neuropathy, no history of IOP exceeding 21 mmHg, and no use of antiglaucoma medications. Except for a cataract and/or glaucoma, no subjects had ocular problems such as clinically detectable ocular inflammation, infection, neuropathies, retinopathies, or maculopathies.Venous blood samples were collected from the antecubital vein using evacuated tubes . The temTo analyze the serum concentrations of reactive oxygen metabolites, antioxidant capacity, and thiol antioxidant capacity, diacron reactive oxygen metabolite (dROM), we performed BAP and sulfhydryl (SH) tests, respectively. For analyses, we used the free radical analyzer system , which include a spectrophotometric device reader and a thermostatically regulated mini-centrifuge. We performed measurements using the measurement kits that were optimized to the FREE Carpe Diem System, according to the manufacturer’s instructions . All anaThe BAP test, measured as its reducing potential against ferric ions, provides an estimate of the global antioxidant capacity of the serum. We measured the intensity of decoloration spectrophotometrically detectable at 505 nm, which occurred when samples are added to a colored solution obtained by mixing a ferric chloride solution and a thiocyanate derivative solution. The measured intensity of decoloration is proportional to the ability of serum to reduce the ferric ions ,31. The The dROM test evaluates an amount of serum hydroperoxides produced by free radicals. The hydroperoxides in the sample mainly react with transition ions liberated from proteins in an acidic medium and are converted to alkoxy and peroxy radicals. These radicals oxidize an additive aromatic amine  and form a relatively stable colored cation radical that is spectrophotometrically detectable at 505 nm ,33. The The SH test estimates the total thiol groups in the biologic samples with a modified Ellman method ,35. The TM MAP Kit , according to the manufacturer’s instructions, as we previously reported [The activities of superoxide dismutase (SOD) 1 and SOD2 in the AH and serum were measured using the multiplex bead immunoassay system, MILLIPLEXreported . SOD1 isreported  and SOD1reported . Based oP values of 0.0167 and 0.0033 for the post-hoc Mann–Whitney U-test were considered significant at 5% and 1%, respectively, based on the Bonferroni correction. Spearman’s correlation test was performed on redox parameters, i.e., SOD1 and SOD2 in AH, and SOD1, SOD2, BAP, dROM, and SH in serum, for which p values of 0.05 were considered significant. The correlations between redox parameters, as mentioned previously, and clinical backgrounds, i.e., age, IOP, and number of topical antiglaucoma medications, were assessed using Spearman’s correlation test, for which p values of 0.05 were considered statistically significant. We performed all statistical analyses using JMP Pro statistical software version 14.2 . All reported p values are two-sided. The data are expressed as the means ± standard deviation (SD) for continuous variables and in numbers and percentage for categorical variables. For the statistical analyses, the decimal BCVA was converted to the logarithm of the minimum angle of resolution (logMAR).For comparisons among the control, POAG, and EXG groups, the differences in continuous data, i.e., age; BCVA; highest IOP; number of topical antiglaucoma medications; and the levels of SOD1, SOD2, BAP, dROM, and SH, were calculated using the Kruskal–Wallis test and the difference in the categorical datum, i.e., sex, was calculated using the G test. For comparisons between pairs of the control, POAG, or EXG groups, we performed the post-hoc Mann–Whitney U-test for continuous data. p = 0.0012) and POAG  groups; The IOP did not differ between the control and POAG groups (p = 0.1042). The number of antiglaucoma medications did not differ between the POAG (2.0 ± 1.5) and EXG (2.9 ± 1.9) groups (p = 0.1884).The demographic subject data, including age, sex, BCVA, IOP, and the number of antiglaucoma medications, among the control, POAG, and EXG groups are shown in p = 0.0251 and 0.0062, respectively) and EXG (p < 0.0001 both comparisons) groups. In serum, the SOD1 levels were significantly higher in patients with EXG compared to controls (p = 0.0137), and the SOD2 levels were significantly higher in patients with POAG (p = 0.0062) and EXG (p < 0.0001) compared to controls. The mean serum BAP values were significantly lower in patients with POAG and EXG compared with controls (p < 0.0001 for both comparisons). The serum dROM and SH concentrations did not differ among the three groups.p = 0.0049). Furthermore, the serum BAP was correlated negatively with both SOD1 (ρ = −0.4485 and p = 0.0020) and SOD2 (ρ = −0.5078 and p = 0.0004) in the AH, and SOD1 in serum (ρ = −0.3524 and p = 0.0190). The dROM and SH levels in the serum were not correlated strongly with the other parameters.The correlations among the redox parameters in the AH and serum are shown in p = 0.0051) and SOD2 (ρ = 0.6596 and p < 0.0001) in the AH and SOD1 in serum (ρ = 0.3075 and p = 0.0423) were correlated positively with the number of antiglaucoma medications. The serum BAP was significantly negatively correlated with the IOP (ρ = −0.4542 and p = 0.0017) and the number of antiglaucoma medications (ρ = −0.6590 and p < 0.0001). A relatively weak but significant correlation was found between the serum dROM and IOP (ρ = −0.3096 and p = 0.0385) and the number of antiglaucoma medications (ρ = −0.3714 and p = 0.0120). In addition, a significant correlation was seen between the serum SH and age (ρ = −0.4898 and p = 0.0006).We compared the redox status both in the AH and serum among patients with POAG and EXG and those without glaucoma and evaluated the relationship among the systemic redox status, intraocular oxidative stress, and clinical backgrounds. Overall, the results suggested three major findings. First, statistically higher SOD species were seen in the AH of patients with POAG and EXG compared with controls. Second, the serum BAP and SOD1 levels were significantly associated with SOD1 in the AH. Third, the serum BAP was correlated negatively with the IOP and the number of antiglaucoma medications. To the best of our knowledge, this is the first study that examined various oxidative stress markers/antioxidative enzymes in serum and aqueous humor by including control, POAG, and EXG subjects, and conducted correlation analyses of various redox parameters and clinical parameters comprehensively.First, our results showed that SOD species in the AH from patients with POAG and EXG were significantly higher than those from controls. Previous reports have found significant increases in SOD activity in the AH in patients with POAG and EXG compared to non-glaucomatous controls ,39. The It was especially noteworthy that the elevations of SOD1 and SOD2 were markedly higher in EXG than POAG. Previous studies have reported that the serum malondialdehyde levels, the end products of lipid peroxides, were significantly higher in patients with EXG than with POAG ,41. Our The second finding is that the decreased serum BAP level, a reliable marker of systemic antioxidative capacity, and increased serum SOD1, a major subtype of SOD species , were co3+) to ferrous ions (Fe2+), and reduced BAP values mean increased oxidative stress in human cells [The third current finding to support this hypothesis is that the serum BAP concentrations were strongly negatively correlated with the IOP and the number of antiglaucoma medications. Moreover, the serum dROM levels were correlated weakly, and the SH levels were not correlated with those factors. Our previous research showed that a serum lower BAP level was correlated with higher IOP and severe visual field damage in patients with open-angle glaucoma ,27. Thosan cells ,31. The an cells . TherefoIt is interesting to note that the SH test measured the total thiol levels in the serum ,35, whicIt is also important to emphasize that the number of antiglaucoma medications was correlated with the SOD species in the AH and the BAP in the serum. Previous reports have shown that topical antiglaucoma medications, especially benzalkonium chloride-preserved eye drops, increased oxidative stress in tear film ,58; therFinally, the current study had limitations that may affect the generalization of our findings. First, our study had the same limitations as any retrospective study in that it was neither controlled nor randomized. Second, the small sample size had insufficient power to detect a significant association among all parameters. Third, the controls were selected from those patients who had undergone cataract surgery, which creates a potential selection bias. Despite these limitations, our study has many strengths, including use of both blood samples and AH, a clinically generalizable sample of patients, and comprehensive assessments of patients’ systemic and ocular redox parameters.In conclusion, the current study suggested that systemic redox status and ocular oxidative stress were associated significantly with EXG and POAG. Further, the systemic redox parameters were correlated strongly with oxidative stress in the AH, and the systemic antioxidant capacity was significantly negatively correlated with the IOP and glaucoma medication score. This study highlights the clinical utility of systemic antioxidant treatment for patients with glaucoma, in particular, elevated IOP-induced glaucoma such as POAG and EXG. The findings warrant further research to explore the underlying mechanisms of the oxidative stress response in glaucomatous patients and to investigate the effectiveness of antioxidative treatment for glaucoma.


P = 0.019, d = 1.083 ( size effect)) and spent more time in the open area of an open field  when young and adult age . Adult female GD offspring showed a reduced level of anxiety , and young female GD offspring showed a deficiency in spatial learning . Adult male GD offspring showed a deficiency in short-term memory . We found an increase in ROS and lipid peroxidation, a disruption in the glutathione status, and decreased activity of catalase and superoxide dismutase , in the cerebral cortex and hippocampus of male and female GD offspring. GD altered metabolism; male offspring of both ages and adult females showed a high level of triglycerides and a lower level of high-density lipoprotein-cholesterol . Young and adult female offspring displayed higher insulin levels . These results suggest that gestational diabetes modifies oxidative stress and cognitive behavior in an age- and sex-dependent manner.Gestational diabetes (GD) has been linked with an increased risk of developing metabolic disorders and behavioral abnormalities in the offspring. Oxidative stress is strongly associated with neurodegeneration and cognitive disruption. In the offspring brains in a GD experimental rat model, increased oxidative stress in the prenatal and postnatal stages was reported. However, long-term alterations to offspring behavior and oxidative stress, caused by changes in the cerebral cortex and hippocampus, remain unclear. In this study, we evaluated the effect of GD on young and adult male and female rat offspring in metabolic parameters, cognitive behavior, and oxidative stress. GD was induced using streptozotocin in dams. Next, the offspring were evaluated at two and six months of age. Anxiety-like behavior was evaluated using the elevated plus maze and open field maze; spatial learning and short-term memory were evaluated using the Morris water maze and radial maze, respectively. We determined oxidative stress biomarkers (reactive oxygen species (ROS), lipid peroxidation and glutathione status) and antioxidant enzymes  in the brain of offspring. We observed that male GD offspring showed a reduced level of anxiety at both ages as they spent less time in the closed arms of the elevated plus maze at adult age (( Gestational diabetes (GD) is a temporary disorder of glucose metabolism during pregnancy, is diagnosed with a fasting glucose level >126 mg/dL, 2-hour glucose 200 mg/dL following a 75 g oral glucose or a casual glucose >200 mg/dL ,4,5,6.In animal models, GD was shown to cause alterations in the neonatal body weight of offspring, as well as alterations in the fetal pancreatic function, morphology, number and size of pancreatic islets, resulting in glucose intolerance or insulin resistance in different organs, such as liver, muscle, and adipose tissue ,8,9. WheStudies on diabetic rat offspring have reported retarded dendritic development and lower expression of the insulin-like growth factor at the embryonic age , a critiSeveral studies have indicated that rats with GD and their offspring are exposed to oxidative stress, reporting increased biomarkers of oxidative stress in the liver, muscle, pancreatic cells, and brain ,18. In tCognitive impairment in the diabetic milieu has been amply recognized in diabetes mellitus (DM) 1 and 2 ,23. In rIn this study we employed a total of twelve Wistar female rats, weighing 280–300 g. Rats were maintained under a normal light/dark cycle (12/12 h) at a temperature of 22 °C in an animal house, with free access to standard rat food and water . All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No.80-23) and the “Norma Oficial Mexicana” regarding the use of experimental animals (NOM-062-ZOO-1999), and approved by the Research Ethics Committee of the Instituto Mexicano del Seguro Social (IMSS) (R-2017-1603-16).n = 6) and gestational diabetic rats . On the ninth day of gestation (mid gestation), diabetes was induced, after 16 h of fasting, by a single intraperitoneal injection of streptozotocin , freshly dissolved in citrate buffer , and GC rats were injected with an equivalent volume of a citrate buffer. To select the STZ dose used in this study we carry out a screening injecting the drug (STZ) at different doses . A 38 mg/kg dose was selected taking into account that elevated glucose levels were maintained in a sustained manner and keeping the pregnancy viable and the viability of fetuses, as well as their development. Gestational diabetes was confirmed by a tail blood sample, and the glucose level was measured using a glucometer  two days later. Only rats that exhibited blood glucose levels between 200 and 350 mg/dL were considered in this study. Glucose levels were monitored throughout pregnancy by taking postprandial blood on days 12, 16, and 20 of pregnancy. At born, all litters were adjusted to 12 pups, maintaining an equal number of males and females. We used two male and two female rats from each litter; after weaning, rats were maintained in groups of four per cage, until the behavioral evaluation. Two-month-old rats (young) and six-month-old rats (adult) male and female offspring from the gestational control (GC) and gestational diabetes (GD) dams; were studied. Behavior tests were applied to 10–12 rats from each group. Next, 5–6 rats were sacrificed to determine oxidative stress biomarkers and the other half of the rats was perfused with formaldehyde solution and reserved for subsequent morphological studies.Female rats were mated with male control rats. Next, vaginal smears were performed over consecutive days to verify the presence of sperm. Once sperm was observed, gestational day 0 had begun, and pregnant rats (dams) were randomly divided into two groups: gestational control rats  and two opposite enclosed arms (50 × 10 × 40 cm); with an open roof. The maze was elevated to a height of 50 cm and lighted by a 60-watt white lamp in the center of the maze. Rats were individually placed in the central area of the maze, and their behavior was recorded for 10 min and analyzed using the Sci-Works software . The time spent in the closed arms was measured.We also evaluated anxiety-like behavior using the open field test. The test consisted of a square arena (60 × 60 cm), divided into eight quadrants  and a central zone. The arena was lighted by a 60-watt white lamp, situated in the center. Similar to the elevated plus maze, the rats were placed individually into the central area, and their behavior was recorded for 10 min and analyzed using the Sci-Works software . The total time spent in the open area  was measured.Next, spatial learning was evaluated using the Morris Water Maze. The maze consisted of a circular pool  filled with water, maintained at 27 ± 2 °C, and dyed dark blue by the addition of gentian violet. The pool contained a circular platform that was placed 2 cm under the water level in a fixed position in one of the four virtual quadrants of the maze during the study. Visual cues were located around the experimental room. Ten different starting positions were placed around the perimeter of the pool. The trial began when a rat was placed in the water facing the wall of the pool at one of the starting points. If the rat failed to find the platform within 60 seconds, it was guided to the platform by the experimenter. Once the rat reached the platform, it was allowed to remain there for 15 seconds.Training consisted of two trials per day . The swimming routes were recorded and analyzed using the Sci-Works software . The escape latency and distance travelled during the 8 days of training, as well as the time spent at each quadrant during the probe trial, were measured.The eight-arm radial maze was employed to evaluate short-term memory. The maze consisted of one central circular arena (30 cm in diameter), from which eight radial arms were extended (60 cm × 10 cm). For this behavioral test, rats were maintained under a food restriction condition  for two weeks, followed by a habituation period: Rats were familiarized with the maze during two sessions. On the first day, they were maintained in the maze for 10 minutes, where free exploration occurred; on the second habituation day, rats were exposed again to the maze for 10 min, with a reward  available in the central area and arms of the maze. On the third day, the training period, the rats were placed in the central area with the eight available arms. The number of visited arms was recorded, and a rat travelling with four feet into the arm was considered a visit. The visited arms were recorded during a maximal period of 10 min, or until the rat had eaten in all arms. Rats performed two 20-minute trials per day over eight consecutive days. Re-entry errors (entries to a previously visited arm), omission errors , and total errors (re-entry errors plus omission errors) were evaluated.Once the behavioral tests were accomplished, rats were sacrificed by decapitation. The brain was dissected, and blood samples were taken.The bilateral hippocampus and cerebral cortex were dissected. Next, tissues were gently chopped. Then, a part of the tissues was homogenized with a buffer , 0.5 % bovine serum albumin (BSA) and 10 mM 3-(N-Morpholino) propanesulfonic acid (MOPS), pH 7.4) to assay the oxidative stress biomarkers , superoxide dismutase (SOD), and catalase activities). The other part was homogenized in 0.9% saline (sodium chloride) and employed for the determination of lipid-peroxidation. Homogenates were stored at −70 °C, until use. The protein content of the homogenates from the cerebral cortex and hippocampus was assayed by a modification of the Biuret procedure  using BSLipid peroxidation was measured by thiobarbituric acid-reactive substances (TBARS) as described by Buege and Aust  with sli2, and 3 mM KH2PO4, pH 7.4) and incubated with 12.5 µM of H2DCFDA for 15 min in an ice bath under constant shaking [ROS were determined using the cell-permeable fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). We resuspended 500 µg of protein from the different tissues in 2 mL of buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 100 mM KCl, 3 mM MgCl shaking . Next, bGlutathione status was determined in accordance with Akerboom and Sies . The tot2O2 to oxygen using a Clark-type oxygen electrode connected to a biological oxygen monitor , according to Jeulin et al. [2O2 was added to the chamber, and the conversion of H2O2 to oxygen was recorded for 2 minutes. Finally, 1.0 mM of sodium azide was added to the chamber. Catalase activity was calculated using bovine catalase as standard. Results are expressed as U × mg of protein. SOD activity was determined using a commercial kit , following the manufacturer’s instructions, and calculated using the SOD from Escherichia coli as standard.Catalase activity was assayed by measuring the conversion of HBlood glucose levels were determined using a commercial blood glucometer , serum lipid  levels were determined enzymatically using reagent kits and following the manufacturer instructions . Serum insulin levels were measured by using a sandwich-type immunoassay (ELISA) commercial kit . t-test. The Morris water maze data were analyzed using a two-way analysis of variance (ANOVA) with the group as the between-subject factor and day as the within-subject factor. Tukey post hoc tests were used if the overall differences were significant (P < 0.05). An analysis of the effect size (d) was conducted if the outcome was statistically significant. Finally, the Pearson correlation coefficients were calculated between behavioral parameters and oxidative stress biomarkers was made.All data were analyzed using Prism . Results are expressed as the mean ± standard error of the mean (SEM), and significance of differences in the mean of body weight, metabolic parameters, anxiety-like behavior, and oxidative stress biomarkers were assessed using an unpaired, a two-tailed Student’s t = 2.944, P = 0.0321). As shown in F  = 84.12, P = 0.0003). We observed as no difference in body weight between rats from the GD and GC groups.A total of six female rats (dams) for group (control and GD) were required to obtain the total male offspring and female offspring for each group and age. Glucose fasting was measured two days after the STZ injection, and a significant increase in fasting glucose in GD, compared with GC female rats was observed , compared with their respective controls. Only young male GD offspring showed a reduced body weight (15%), and the reduction remained into adulthood .t = 2.42, P = 0.0193, d = 0.992, and t = 2.53, P = 0.0199, d = 1.083, respectively), suggesting that GD male and female offspring rats experienced a reduced level of anxiety in adulthood compared with the normoglycemic offspring. In the open field, the time spend in the center (open area) is considered an anxiety level index (rats spending more time in this open area are considered less anxious). As shown in To determine the effect of GD on the anxiety of the offspring, the performance of rats in the plus maze and open field were evaluated. In the plus maze, the time that rats spent in the closed arms is considered an anxiety index. Male and female GD offspring spent a similar amount of time in the closed arm, compared with their respective control group when young a, b. BotF  = 1.07, P = 0.32 and F  = 0.41, P = 0.53 for the young and adult age groups, respectively). In the probe trial (ninth day), rats from both groups (GD and C groups) spent more time at the platform position (target) instead of the other maze quadrants, and the performance of the groups was similar, as shown in F  = 6.71, P = 0.029, d = 3.207), and paired comparisons showed significantly longer distances at days 7 and 8 for the GD compared with the female C group . Accordingly, with deficient learning, in the probe trial (ninth day), GD female rats spent the same amount of time in all zones of the maze, whereas the rats from the C group searched the platform for the target quadrant, as shown in To determine if GD disrupts spatial learning, the performance of young and adult  offspring of GD rats was assessed using the Morris water maze test for eight days. No statistical differences were found in terms of the swimming distances of male rats of any age during the training days compared with the control group  = 1.72, P = 0.22 and F  = 0.26, P = 0.61). They efficiently performed the task, making fewer errors during the training days . We found a significant difference between adult male offspring of GD rats and the control group in the number of re-entry errors  = 2.61, P = 0.017, d = 1.695), and a paired test revealed that a higher number of re-entry errors were committed by GD group rats on days 7 and 8 . These results show a short-term memory deficiency. Next, to evaluate the effect of GD on the spatial working memory of offspring, we used an eight-arm, eight-day radial maze test . Young mF  = 2.15, P = 0.050) due to the reduction of the number of omissions, since the number of re-entry errors did not show significant differences compared with the control, and they efficiently performed the task during the training days.Female GD offspring showed a significant decrease in the number of total errors in adulthood from days 3 to day 5 , whereas the female GD offspring showed a higher level in this parameter only in adulthood . The HDL−cholesterol level was low in young and adult male GD offspring , and the insulin serum level was low in young male GD offspring . This change did not remain into adulthood. Similar to young GD offspring, we observed a low level of HDL−cholesterol in young and adult female offspring  and a higher insulin level at both ages .To determine the impact of gestational diabetes on metabolic parameters, the glucose, insulin, cholesterol, and triglycerides in the serum of young and adult male and female offspring were measured . No statP < 0.05 were considered, as shown in Finally, we calculated the Pearson correlation coefficients between the parameters of the behavioral tests and oxidative stress biomarkers. Only correlations that were significant at In this study, we examined the effects of gestational diabetes on oxidative stress and their relationship with behavioral impairment linked to the hippocampus and cerebral cortex function in young and adult rat offspring. Adverse environment exposure during embryonic and early postnatal development may lead to a risk of developing metabolic disease in later life ,31,32,33Gestational diabetes modifies the offspring’s body weight at birth in a maternal glucose-dependent manner. Our results only indicated a lower body weight at birth (microsomia) in male offspring of GD rats, and this change remained during the weaning, juvenile, and adult stages. This weight reduction at birth could be due to the high blood glucose concentration in the intrauterine milieu during gestational diabetes, since it was reported to induce pancreatic hypertrophy due to the over-stimulation of pancreatic β cells, preventing the secretion of insulin and decreasing anabolism and, subsequently, neonatal microsomia ,34,35,36Our most striking findings are the cognitive changes in the offspring exposed to gestational diabetes. Several studies have reported disturbances in the anxiety-like behavior of gestational diabetes offspring. Ramanathan et al.  showed tCurrently, few studies have assessed the effect of gestational diabetes on cognitive performance. In this study, we evaluated spatial learning and short-term memory. We obtained findings of age- and sex-dependency. Whereas the male offspring of gestational diabetic rats at young and adult ages did not show disruptions in spatial learning, the female progeny showed an impairment of cognitive performance at the juvenile age. These results are in accordance with those reported by Kinney et al. , who fou2+ homeostasis, and impairment of glutamate transport and mitochondrial function in synapses [Optimal brain function is dependent on cellular redox homeostasis, and neurodegenerative diseases, cognitive decline, and cerebral dysfunction are closely related to oxidative stress. At the embryonic stage, under hyperglycemia, a link with oxidative stress has been reported in the intrauterine milieu, and ROS reduction contributes to the decrease in oxidative damage to developing embryos in rats with gestational diabetes ,47. For synapses . These results suggest that the cerebral cortex and hippocampus are susceptible to oxidative damage and are therefore liable to present an altered function. The antioxidant system was disturbed in male and female GD offspring in the cerebral cortex and hippocampus at the juvenile age, which remained into adulthood. Oxidative stress resulting from a hyperglycemic intrauterine milieu increased the oxidative damage and alteration in the antioxidant defense during fetal  and neonAccording to many studies that support the link between exposure to an adverse intrauterine milieu, including hyperglycemia, and a subsequent risk of developing metabolic diseases ,50,51,52In summary, these results suggest that gestational diabetes modifies oxidative stress and cognitive behavior in an age- and sex-dependent manner. However, the relationship between oxidative stress and cognitive performance is complex. Therefore, the evaluation of other possible mediators, such as the impact of oxidative stress and the inflammatory response to neuronal function and neuronal morphology, is necessary to elucidate the possible underlying mechanisms associated with behavioral/cognitive alterations and their possible effect across generations.Gestational diabetes modifies the oxidative stress in the cerebral cortex and hippocampus as well as cognitive behavior in an age- and sex-dependent manner.