In this study, we investigated subjective geriatric complaints (SGCs) as conditions regarding health concerns in community-dwelling older people and analyzed their frequencies with aging and relationships with other factors.This cross-sectional study enrolled 10,434 older people living in a community with a representative aging population in Japan. A questionnaire was sent by mail to those who had not applied for formal care needs certification. The presence of and concern for symptoms common in old age were asked as SGCs, as were physical function levels, multimorbidity, and depression. Categorical principal component analysis (CATPCA) of the symptoms was performed, and the association between the obtained components and other factors was analyzed.The mean age of the participants was 73.7 ± 6.1 years, and 52.5% were women. On average, they had 1.72 ± 1.57 SGCs, which showed a gradual increase with age. The results of the CATPCA revealed four components: SGC1, excretory/circulatory/swallowing complaints; SGC2, audiovisual complaints; SGC3, neurological complaints; and SGC4, musculoskeletal complaints. All SGC components were independently associated with physical function, multimorbidity, and depression.Each SGC showed various frequencies and differences along with aging, and SGCs were classified into four components that were thought to share a common background. These findings could contribute to the planning of better health management strategies for older people. The aging of the population and advances in medical treatment have resulted in an increasing number of older people with concurrent multiple diseases and conditions. The co-occurrence of multiple chronic or acute diseases in an individual is known as multimorbidity, and this has recently become an important issue . In addiThe concept of geriatric syndromes is very important in geriatric medicine, and their presence has been shown to be a prognostic factor in emergency hospitalization among the aged –9. HowevRecently, frailty has been noted as an intermittent condition between robustness and disability and a risk factor for geriatric syndromes –16. It iAlthough the health consequences associated with aging mentioned above are widely known, many older adults continue to live independently in their community while being concerned about the various symptoms associated with aging. These symptoms, designated here as subjective geriatric complaints (SGCs), consist of common age-related symptoms that community-dwelling older people experience in daily life. While some of these may overlap with those of geriatric syndromes, e.g., vision impairment and urinary incontinence, we assume that SGCs can be distinguished from geriatric syndromes from their clinical characteristics. For example, these symptoms remain relatively stable and would not have a major influence on the independence of older people. It has also been suggested that the aged may perceive health problems at a prodromal stage , 25. TheAs part of the TOyoake Integrated Care Study (TOICS), a questionnaire was sent to 14,850 people aged 65 years or older living in Toyoake city (89.2% of the older people in the city) in a suburb of metropolitan Nagoya city, Japan . No partThe questionnaire was composed of items on age, sex, family composition, body mass index, physical function, multimorbidity, and depressive mood. With regard to SGCs, in the questionnaire, we asked, “Which of the following symptoms may interfere with your daily life?” and listed 14 symptoms: dizziness, headache, abdominal pain, insomnia, urination disorder (pollakiuria and/or urinary incontinence), defecation disorder (constipation and/or diarrhea), vision impairment, hearing loss, appetite loss, low back pain, arthralgia (including tingling), dysphagia (including choking), shortness of breath, and edema. These lists of symptoms were constructed according to existing lists of geriatric syndromes and related publications , 28–30. Regarding physical function, we asked the following three questions related to physical ability from the Kihon Checklist (KCL) : (1) “DoThe participants’ characteristics, SGC score, physical function score, multimorbidity score, and depression status were summarized using basic descriptive statistics. The mean, standard deviation (SD), and proportion were used as appropriate. Differences in basic characteristics among age groups were compared using one-way analysis of variance for continuous variables and chi-squared tests for categorical variables. To show aging-related changes in 13 SGCs, the frequency (%) of symptoms was indicated based on six age groups . The difference in frequencies in younger age groups  and older age groups  was shown based on the ratio of frequencies in both groups.P value of < .05 was considered significant.Categorical principal components analysis (CATPCA) with varimax rotation was performed to examine the factor structure of the SGCs since the responses to the questionnaire were nominal. To select the optimal number of components, the Kaiser criterion  was employed. Items with factor loading >.30 were retained. Multiple regression analysis was conducted to evaluate the association between each SGC component and physical function, multimorbidity, and depressive mood. All statistical analyses were carried out using IBM SPSS for Windows . A two-tailed The mean age of the participants was 73.7 ± 6.1 years, and 52.5% were women. The physical function scores for each age group (in 5-year increments) decreased with age (2.46 ± 0.84 in the 65–69 to 1.43 ± 1.10 in the 90 years and older group). On the other hand, the multimorbidity score increased with age (1.16 ± 1.13 in the 65–69 to 1.90 ± 1.36 in the 90 years and older group), as did the SGC score (1.47 ± 1.39 in the 65–69 to 2.26 ± 1.78 in the 90 years and older group) and proportion of higher numbers of SGCs. On average, 21% of the participants did not have subjective complaints. Rates of depression did not change across age groups .The frequencies of SGCs are indicated in Then, CATPCA was performed to examine the factor structure of the SGCs. CATPCA based on the responses of 13 SGCs in 10,434 individuals aged 65 years and older revealed four components: SGC1, excretory/circulatory/swallowing complaints; SGC2, audiovisual complaints; SGC3, neurological complaints; and SGC4, musculoskeletal complaints . SGC1 inMultiple regression analysis using the number of SGCs in individuals in each of the four components as the dependent variable and physical function scores, multimorbidity scores, and depression as independent variables was performed to identify associations. The results revealed that all four SGC components were significantly independently associated with physical function scores, multimorbidity scores, and depression after adjusting for age and gender .In this study, we characterized the symptoms of health concerns as SGCs among older people living in the community who were not receiving long-term nursing care. Changes in the frequency of SGCs with aging revealed SGCs with a low frequency of complaints in all age groups, SGCs with a steep increase with age, and SGCs with a high frequency of complaints that did not show a noticeable change with age. CATPCA was performed to examine the relationships between SGCs and physical function, multimorbidity, and depressive mood, and the following four components were obtained: SGC1, excretory/circulatory/swallowing complaints; SGC2, audiovisual complaints; SGC3, neurological complaints; and SGC4, musculoskeletal complaints. Each component was associated with multimorbidity, physical function, and depressive mood.Geriatric syndromes are well-known conditions that occur with aging. The classical geriatric syndromes are associated with the so-called “giants of old age” , 6, 11. Multimorbidity and related polypharmacy may also be a major health challenge for the aged , 34–36. In recent years, the concept of frailty has been proposed for the early detection of conditions leading to the need for nursing care in old age, and preventive interventions have been undertaken , 31, 40.Based on the results of the present study and related previous studies regarding geriatrics , 35, 40,Although we used three items of the KCL as the physical function score in this study, it was assumed to be associated with frailty . DepressIn Japan, the aging rate (age 65 years or over) has reached nearly 30%, and the average life expectancy for both men and women is over 80 years, making it an important issue to determine what health issues arise among the aged and when. As the global population is also aging, we believe that the situation in Japan is also important internationally. In this study, conducted jointly by medical professionals and policy makers, we were aware of the need to raise awareness and intervene at the right time to address such issues among the local population.This study had several limitations. First, it was a cross-sectional survey, and we were unable to show an association between SGCs and longitudinal outcomes. In the future, it will be desirable to clarify the significance of SGCs by conducting a longitudinal analysis of the QOL associated with SGCs. Second, since this was a questionnaire-based survey, physical measurements and examinations were not performed. Future cross-sectional analyses and longitudinal studies of SGCs incorporating the actual measurement of some items would be important for a more detailed analysis. Third, we did not examine how the symptoms described as SGCs are related to actual diseases. For example, it would be desirable to investigate in detail the relationship between each SGC and individual diseases, such as whether complaints of visual impairment occur in presbyopia, cataracts, or age-related macular degeneration.In conclusion, analyzing the complaints and health concerns of older people living in the community as SGCs may help to plan more effective interventions from an earlier stage and lead to the prevention of multimorbidity, frailty, and geriatric syndromes.


Multimorbidity, the co-occurrence of two or more chronic conditions, is common among older adults and is associated with decreased quality of life, greater disability, and increased mortality. Yet, the association of multimorbidity with pain, another significant contributor to decreased quality of life, has not been widely studied. This is especially understudied among very old (aged ≥ 80) Mexican Americans, a fast-growing segment of the United States (US) population.To assess the association of multimorbidity with pain in very old Mexican Americans, over six years of follow-up.We used data from Waves 7 (2010/2011) to 9 (2015/2016) of the Hispanic Established Populations for the Epidemiologic Study of the Elderly, a longitudinal study of older Mexican Americans residing in the Southwestern US. Multimorbidity was defined as reporting two or more chronic health conditions. Pain was defined as (1) pain on weight-bearing, (2) pain in back, hips, knees, ankles/feet, legs, entire body, or two or more locations, and (3) pain that limits daily activities. We use generalized estimation equations to estimate the odds ratio of pain as a function of multimorbidity over 6 years.n = 841), 77.3% of participants had multimorbidity. Those with multimorbidity had greater odds  of reporting pain on weight-bearing over time, compared to those without multimorbidity. Also, those with multimorbidity had 2.12 times the odds of reporting pain that limited their daily activities  compared to those without multimorbidity. Lastly, those with multimorbidity had higher odds of reporting pain in their back, knee, ankles/feet, legs, hips, entire body, or two or more locations, compared to those without multimorbidity.At baseline (Those with multimorbidity consistently had higher odds of all types of pain, highlighting the need for early management of pain among those with multiple chronic conditions and complex health needs. This is especially important among very old Mexican Americans, who have a high burden of chronic health conditions. The worldwide population is rapidly aging. The proportion of the population aged 60 and older is expected to nearly double, from 12% in 2015 to 22% in 2050 , 4. ThisPain is common among adults in the US, particularly among older adults and women. In 2019, among those aged 65 and older, 30.8% had chronic pain and 11.8% had high impact pain, defined as chronic pain that limits life or work activities , greaterMultimorbidity, defined by the World Health Organization (WHO) as “the coexistence of two or more chronic conditions in the same individual,” is common among older adults and women . A literMexican Americans are an important group to study because of the previously documented Hispanic Paradox , 29, whiPrevious work examining racial/ethnic differences among middle-aged adults in the US found that, while Hispanic adults had fewer chronic diseases at baseline, they accumulated chronic diseases 1.5% faster than non-Hispanic Whites . Older MThe objective of this study was to assess the association of multimorbidity with pain over 6 years of follow-up. We examined pain on weight-bearing, pain that limits daily activities, and pain site. We hypothesize that multimorbidity will be associated with greater pain among Mexican Americans aged 80 and older.n = 3,050). In 2004/05 a new cohort of 902 participants aged 75 years and older were added to the 1,167 participants of the original cohort who were 75 years and older . Bilingual interviewers were trained to gather information on socio-demographics, health conditions, and psycho-social characteristics of respondents in their language of choice every 2 or 3 years. Detailed information on the H-EPESE is publicly available at the National Archive of Computerized Data on Aging (We used data from Waves 7 (2010/2011), 8 (2012/2013), and 9 (2015/2016) of the Hispanic Established Population for the Epidemiologic Study of the Elderly (H-EPESE), a longitudinal study of older Mexican Americans residing in the Southwestern US , 34. Theon Aging .n = 52), those with missing information on pain at baseline (n = 158), and those with missing information on covariates (n = 27). Our final analytical sample was 841. Those excluded from our analysis had fewer years of education on average, lower Mini-Mental State Examination Score (MMSE) scores, and more often reported high depressive symptoms, pain on weight-bearing, and pain that limited their daily activities . Those who respond yes were then asked, “where was this pain”  and “does this bodily pain last for four or more weeks?” (yes/no). We created a pain site score variable which was a sum of the number of locations where pain was reported (range: 0-6). Participants were also asked: “in the past month, how much has this pain or discomfort restricted your daily activities” and “in the past month, how much has this pain and discomfort kept you from getting a good night's sleep” with “a lot,” “some,” or “not at all” as response options. Participants who responded “a lot” or “some” were considered to have pain that limited their daily activities or pain that interfered with their sleep, respectively. Lastly, participants were asked “have you ever seen a doctor about your pain” (yes/no) and “do you take any medication for your pain” (yes/no). These specific pain questions were assessed at Waves 7 through 9 of the H-EPESE, except for pain lasting longer than 4 weeks, which was assessed only at Waves 7 and 8.Sociodemographic and health characteristics were included as covariates in these analyses. These included age, sex , marital status , education, nativity , language of interview , MMSE score , and higt-tests were used to test the baseline descriptive characteristics of participants by multimorbidity status. We used general estimation equations to estimate the odds ratio (OR) and 95% Confidence Interval (CI) of (1) pain on weight-bearing; (2) pain location; and (3) pain that limits daily activities, over 6 years as a function of multimorbidity. We conducted a sensitivity analysis using general estimation equations to estimate pain location as a function of individual diseases included in our definition of multimorbidity. To understand how our findings may have been impacted by the exclusion of those with lower MMSE scores, we conducted a second sensitivity analysis where we examined the descriptive statistics of pain by cognitive impairment (MMSE < 21)  , 39. Forn = 841) was 85.8 years old on average [standard deviation (SD): 3.9] and 64.1% were female (p < 0.05). Those with multimorbidity had higher reported rates of all health conditions compared to those without multimorbidity. Hypertension, arthritis, and diabetes were the most common conditions reported among both groups. Among those with multimorbidity, the most common co-occurring conditions were hypertension and arthritis, hypertension and diabetes, arthritis and diabetes, arthritis and osteoporosis, and hypertension and heart failure.Overall, our sample  (p < 0.05). Among those with multimorbidity, 28.6% reported pain at two or more locations, compared to 12.6% of those without multimorbidity (p < 0.05). Those with multimorbidity more frequently reported pain that limited their daily activities a lot (19.8% vs. 6.8%) or somewhat (25.7% vs. 19.9%) (p < 0.05). Among those reporting pain, there was no difference in pain lasting longer than 4 weeks, pain interfering with sleep, reporting doctor visits for pain, or taking medication for pain, based on multimorbidity status. Overall, most participants reported pain lasting longer than 4 weeks (83.0%), seeing a doctor for pain (89.3%), and taking medication for pain (79.1%). Participants with multimorbidity more often reported pain on weight-bearing ( < 0.05) . The mosThose with multimorbidity had greater odds  of reporting pain on weight-bearing and pain that limited their daily activities  over time, compared to those without multimorbidity . Those wn = 841), 39.1% of participants had MMSE < 21 while 60.9% had an MMSE ≥ 21. We found that, among those with MMSE < 21, 51.1% reported pain on weight-bearing while 44.0% of those with MMSE ≥ 21 reported pain on weight-bearing (p = 0.043). In our third sensitivity analysis, among only those with MMSE ≥ 21 in our analytical sample (n = 512), we found consistent results where multimorbidity was associated with pain on weight-bearing  and pain that limits daily activities . The results of our fourth sensitivity analysis, where we included all H-EPESE participants , regardless of complete baseline data, were fairly consistent but more conservative for pain on weight-bearing  and pain that limits daily activities .We conducted a sensitivity analysis, where we included each disease individually . Those wIn our study of community-dwelling Mexican Americans aged 80 and over, we examined the association of multimorbidity with pain, pain location, and pain that limited daily activities over 6 years of follow up. We found that about 77% of participants had multimorbidity; of these, over half (51.7%) reported pain on weight-bearing. Multimorbidity was associated with higher odds of pain on weight-bearing, pain that limited their daily activities in the past month, pain at each pain location, and overlapping pain at two or more locations.Our findings are consistent with previous work that found a link between pain and multimorbidity –25, 27. The results presented in this study build on the literature on the association between multimorbidity and pain, as few studies on multimorbidity have examined locations of pain, how pain interferes with daily activities, and overlapping pain sites. We found that, among those with multimorbidity, almost 30% reported pain at two locations, compared to about 13% of those without multimorbidity. When we examined overlapping knee, back, and multisite pain by multimorbidity status, we found that those with multimorbidity reported knee, back, and multisite pain over twice as often as those without multimorbidity. This result is consistent with previous work using data from the UK data bank among participants aged 37-73 who documented a dose-response relationship between number of long-term chronic conditions and pain .The intersection of pain and multimorbidity has clinical implications. Most evidence-based guidelines have limited applicability to individuals with co-morbid chronic conditions , 40. BecThe mechanisms underlying pain and multimorbidity are not well understood. However, a direct potential mechanism may be dysregulation of physiological mechanisms and accumulated disruptions across multiple systems, which is associated with increasing age and the presence of multimorbid diseases and frailty syndrome , 45. OthOur findings should be considered along with some limitations. First, selection bias may have occurred through the inclusion of individuals aged 80 and older at baseline. This may have resulted in a more robust, healthier sample, and may have underestimated the true relationship between multimorbidity and pain. Second, our findings may be susceptible to recall bias due to our older population and self-reported health conditions. Third, we may have underestimated the relationship between multimorbidity and pain, as those who were excluded had lower MMSE scores and more often reported pain. Fourth, since our sample was restricted to Mexican Americans living in the Southwestern US, our findings may be not generalizable to the Hispanic population in the US. The Hispanic population is heterogeneous and pain burden differs by country of origin, as older adults from different nationalities may have different health profiles and life course experiences that influence their development of pain , 47. DesIn this sample of very old community-dwelling Mexican Americans, those with multimorbidity consistently had higher odds of pain, including pain at two or more locations, highlighting the need for early management of pain among those with multiple chronic conditions and complex health needs. This is especially important among very old Mexican Americans, who have a high burden of chronic health conditions. Very old Mexican Americans are a growing, yet understudied, group, and studies on pain and multimorbidity are mostly based on non-Hispanic White cohorts. Future work is needed to understand the mechanisms underlying overlapping pain conditions and multimorbidity, especially in the context of the syndemic and intersectionality frameworks –18.https://www.icpsr.umich.edu/web/NACDA/series/546].Publicly available datasets were analyzed in this study. This data can be found at: National Archive of Computerized Data on Aging [The studies involving human participants were reviewed and approved by Institutional Review Board at the University of Texas Medical Branch (IRB # 92-85). The patients/participants provided their written or oral consent to participate in this study.SAM was responsible for study design, analyses, interpretation of the results, and writing of the manuscript. SAS was involved in study design, analyses, interpretation of the results, and editing of the manuscript. YFK, DSL, KSM, and MAR were responsible for study design, interpretation of the results, and editing of the manuscript. All authors contributed to the article and approved the submitted version.SAM was supported by a Research Career Development Award  from the National Institutes of Health/Office of the Director (OD)/National Institute of Allergy and Infectious Diseases (NIAID), and Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD). The Hispanic Established Populations for Epidemiological Studies of the Elderly (H-EPESE) was funded by the National Institutes of Health, National Institute on Aging . YFK and MAR were supported by the National Institute on Drug Abuse . SA was supported by the National Institute on Aging . DSL was supported by the National Institutes of Health (NIH) and National Institute on Aging, Grant # P30 AG059301; and Cancer Prevention and Research Institute of Texas, Grant # RP210130. This work was also supported by the Texas Resource Center on Minority Aging Research  and the Claude D. Pepper Older Americans Independence Center .The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The Automated Anatomical Labeling (AAL) 116 atlas was used to extract the histogram features of the regions of interest in the brain. Machine learning methods were used in the primary set for data dimensionality reduction, feature selection, model construction, and model performance evaluation. The model performance was further validated in the validation set. After feature data dimension reduction and feature selection, 23 of a total of 1,276 features were entered in the model. The brain regions of the selected features included the frontal, temporal, parietal, occipital, and limbic lobes, as well as the cerebellum and the thalamus. In the primary set, the area under the curve (AUC) of the model was 0.974, the sensitivity was 93.8%, the specificity was 90.6%, and the accuracy was 93.8%. In the validation set, the AUC, sensitivity, specificity, and accuracy were 0.980, 90.9%, 88.9%, and 90.0%, respectively. ALFF-based histogram analysis can be used to classify patients with PD and HCs and to effectively identify abnormal brain function regions in PD patients.This study aimed to investigate the value of amplitude of low-frequency fluctuation (ALFF)-based histogram analysis in the diagnosis of Parkinson’s disease (PD) and to investigate the regions of the most important discriminative features and their contribution to classification discrimination. Patients with PD ( Parkinson’s disease (PD) is one of the most common clinically progressive neurodegenerative diseases worldwide, with prevalence second only to Alzheimer’s disease, and it affects more than 10 million people worldwide  T1-weighted structure images and the rs-fMRI data of each participant were collected with a 3T Siemens MRI scanner . The parameters were as follows , spatial smoothing using a 6 × 6 × 6-mm3 full-width half-maximum Gaussian kernel, high-pass filtering (0.01–0.08 Hz), and linear drift removal. The ALFF calculation and mean standardization were further performed on the preprocessed rs-fMRI data.The data obtained in this study are the mean ALFF images. The data had already been preprocessed and the mean ALFF had been calculated. This processing is a standardized preprocessing step. The detailed mean ALFF preprocessing and calculation procedure can be found in a previous study  cross-validation for feature selection. We also reported the AUC, accuracy, sensitivity, specificity, and the 95% CI to evaluate the performance of the model.2 test was used to assess the sex differences between patients with PD and the HCs, and the independent samples t-test was used to estimate the differences between PD patients and HCs in age, years of education, and MMSE scores. LASSO and ROC analyses used the “glmnt” and “pROC” software packages in R software. P < 0.05 was considered statistically significant.The statistical analysis of this study was conducted using MATLAB 2012b  and R version 3.6.1In the primary and validation sets, there was no statistically significant difference between the HCs and PD patients in terms of sex, age, years of education, and MMSE scores .t-test after Z-standardization showed that 236 of the 1,276 features had intergroup differences (P < 0.05); the multicollinearity variables  were deleted for the remaining 236 features. Finally, the remaining 129 features (λ as the feature selection criteria. When the MSE was minimum (λ = 0.069), there were 23 nonzero features mean, left inferior parietal gyrus mean, left paracentral lobule mean, left thalamus mean, left inferior cerebellum_9 (Cerebelum9L)minimum, left inferior occipital gyrus maximum, right inferior temporal gyrus maximum, left superior cerebellum_6 (Cerebelum6L)maximum, right supplementary motor area (Supp_MotorAreaR)standard deviation, left inferior occipital gyrus standard deviation, right precuneus (Precuneus_R)median, left parahippocampal gyrus skewness, right parahippocampal gyrus skewness, right amygdala skewness, left middle occipital gyrus skewness, right inferior parietal gyrus skewness, left frontal middle gyrus kurtosis, left superior frontal gyrus 10th percentile, left supplementary motor area (Supp_MotorAreaL)10th percentile, left inferior parietal gyrus 10th percentile, right inferior parietal gyrus 10th percentile, and the right thalamus 10th percentile  in the ROI defined by the brain atlas (Dai et al., t-test on the standardized data to select the features that were significantly different between groups for subsequent analysis. Then, we applied previous research data processing methods (Mo et al., Radiomics can extract a large number of quantitative features; however, its data dimensionality is too high when compared with the sample size of most studies, making it is easy to fall into a “curse of dimensionality,” thus causing the model to overfit. Hence, the features must be selected for dimensionality reduction to obtain the most valuable features in order to improve the reliability and accuracy of the model (Gu et al., Previous studies (Varoquaux, In this study, the discriminative brain areas that can be used for PD diagnosis are located in the frontal, temporal, parietal, occipital, and limbic lobes, as well as in the cerebellum and the thalamus, which are consistent with the results of previous PD studies (Szewczyk-Krolikowski et al., This study has some limitations. Firstly, as obtaining a large dataset is difficult in neuroimaging, where data acquisition is costly, the sample size of this study is relatively limited, but compared with the sample sizes of previous intergroup comparison studies (Lei et al., In conclusion, this study used rs-fMRI data to extract the histogram features of brain regions based on brain atlases and used machine learning analysis to build a PD classification model. The study showed that this method can classify PD patients and HCs well and effectively identify PD patients’ regions of abnormal brain function; furthermore, it can assist in the early diagnosis of PD and provide a means for PD mechanism research and clinical therapeutic efficacy evaluation.The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Medical Research Ethical Committee of Nanjing Brain Hospital. The ethics committee waived the requirement of written informed consent for participation.DS conducted the experiment, performed the data processing and analysis, and wrote the manuscript. HZ, GW, and SW collected the data and performed the data processing and analysis. KR supervised the whole study, including experiments and manuscript writing. 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.


This study examined the 3-month rate of bullying experience, associated factors, and measure the relationships between bullying experience with health-related quality of life and different mental disorders among secondary school students. We performed a cross-sectional study in four secondary schools in Hanoi, Vietnam. Bullying experience was evaluated by using questions about eighteen specific-bullying behaviors. EuroQol-5 dimensions-5 levels (EQ-5D-5L) and Depression, Anxiety, and Stress Scale– 21 items (DASS-21) were used to measure health-related quality of life (HRQOL) and mental health of participants, respectively. Among 712 secondary school students, the 3-month prevalence of physical, social aggression, verbal, and sexual bullying experience were 8.4%; 31.2%; 11.9%, and 2.7%, respectively. Being bullied were negatively associated with levels of classmates and family support, as well as levels of school security. Being overweight or obese was related to a higher likelihood of suffering social aggression compared to normal BMI. Being bullied was significantly associated with the decrement of HRQOL, and the increased risk of depression, anxiety, and stress among adolescents. Findings of this study suggested that holistic approaches involving family, peers, and schools, along with enhancing school security, are potential approaches to reduce the impact of bullying on adolescents’ life and well-being. Bullying among children is a serious global health problem given its profound physical and psychological consequences on both bullies and victims . BullyinThe prevalence of bullying victimization is diverse across nations. A multi-country cross-sectional study showed that the rate of bullying ranged from 2.4% to 31.9% among boys and from 1.5% to 34.4% among girls . PhysicaIn Vietnam, a previous study suggested that approximately one-third of students at secondary and high schools experienced as victims, bullies, or bully-victims at both times . Positivth to 9th grade); 2) studying in selected schools and 3) accepting to be the study participant. Informed consents were given to the principals of selected schools, teachers, students, and their parents/guardians before collecting data.From January to September 2020, we performed a cross-sectional study at four secondary schools in Hanoi, a metropolis of Vietnam. We recruited students who met the following inclusion criteria: 1) aged from 11 to 14  α = 0.05, expected population proportion p = 47.5% , relativIn this study, we anonymously collected students’ data by using a self-administered structured questionnaire. Given the sensitive content of the question, this questionnaire was developed under the instruction of experts in child maltreatment and bullying. The questionnaire was carefully piloted among children and adolescents to ensure that the content of the questions was understandable and logical. We also revised all texts and language errors in the questionnaire. The final version of the questionnaire was approved by the principals of selected schools. Before administration, the principal investigator briefly introduced the purposes of the study and guided students in filling the questionnaire. In each class, the questionnaire was distributed to the students during non-teaching hours and then collected by the researcher. Students spent 15 to 20 minutes to complete the survey.2) = height/weight2). Students were classified into three groups: underweight (< 18.5 kg/m2), normal (18.5- < 25.0 kg/m2) and overweight/obesity (≥25.0 kg/m2).We included specific questions to measure socio-demographic characteristics including age, gender , type of family (nuclear/others), height, and weight. We then calculated the body mass index by using height and weight information Threatened with a weapon Locked in the classroom, toilet or other roomsRobbed moneyNone of themIn the last three months, have you ever been:Disrespected and refused to listen to your opinionTaken control by someone and made you do what they want?Boycotted by someone or incited others to boycott or isolate you?Bullied or scared you?Forced or seduced you to participate in destructive and anti-social behaviours?Put pressure on you to achieve results beyond your capabilities?None of themIn the last three months, have someone everTalked profanely or looked down on yourself and my family by someone, or made fun of, mimicked, or imitated your behaviours?Commented or told a story in order to insult or offend / ridicule you?Discussed, gossiped, or spread bad rumours about youPretended to be you on the internet and put private information about you online?None of themIn the last three months, have someone everPulled, taken off, tugged your skirt / shirt / pants?Touched a sensitive part of your body?Forced you to have sex?Sent text messages or emails asking you to have sex that you did not want?None of themIn the last three months, have someone ever:Participants selected any options of the above questions were categorized as “Having experience”. For question 1, we aimed to investigate the physical bullying victimization, while for questions 2, 3, 4, we aimed to examine the social aggression, verbal bullying, and sexual bullying experience, respectively. Students who did not select any of them or select “None of them” were classified as the “No experience”.Students were asked seven self-reported questions about perceived levels of support from family (item 1 and 2), classmates (item 3 and 4), and teachers . This approach has been used previously :My parents do not understand me or care about my feelings.My parents do not listen to me or not pay attention to the problems I have.My classmates are very friendly.Classmates respect me and listen to my opinion.My teachers help me when I’m sad or having problems.My teachers take care of me and support me in achieving the best results.My teachers respect myself and listen to me.There were five levels of response for each statement: 1 = Totally disagree; 2 = Slightly agree; 3 = Somewhat agree; 4 = Mostly agree, and 5 = Totally agree. We reversed scores of questions 1 and 2 when calculating the total score. The total score of each domain was calculated by summing scores of all items in the domain and divided by the number of items in each domain. The score ranged from 1 to 5, which a higher score indicated higher levels of support. Cronbach’s alpha of these items was 0.820, suggesting acceptable internal consistency reliability. In addition, we asked students to rate levels of school security with an 11-point scale from 0 “Complete unsafety” to 10 “Complete safety”.HRQOL of students was assessed by utilizing the Vietnamese version of EuroQol-5 dimensions-5 levels (EQ-5D-5L) . This scThe Depression, Anxiety, and Stress Scale—21 Items (DASS-21) was used to evaluate the depression, anxiety, and stress conditions of students in the last seven days . This scData analysis was conducted using the Stata 15.0 software. P-value < 0.05 was used to detect statistical significance. Missing data were handled using the listwise deletion method. Variance inflation factor (VIF) was tested to examine the collinearity in regression models.The 3-month prevalence of different types of bullying  were described in general and according to different socio-demographic characteristics. We also defined “poly-victimization” as students who suffered from two or more forms of bullying . The ChiEQ-5D index and the rate of depression, anxiety, and stress among students were described according to types of bullying victimization. The difference between victims and non-victims was examined using the Mann-Whitney test (for EQ-5D index) and the Chi-squared test . Associations between the EQ-5D index and types of bullying victimization as well as the number of forms of bullying experienced were identified by using the multivariate Tobit regression. Meanwhile, relationships between depression, anxiety, and stress with types of bullying victimization as well as the number of bullying forms experienced, were determined by using the multivariate Logistic regression. These models were adjusted for prior-defined-sociodemographic characteristics, levels of school security, and levels of parents, classmates, and teacher support.Given that bullying experience was the sensitive information, before implementing the survey, we contacted and send an information package to the principals of selected schools, teachers, students, and their parents/guardians. Each package contained information about the study purposes, designs, inclusion criteria, and potential benefits and harms when participating in the study. We also underlined that students’ participation was not mandate, and they could withdraw to the study anytime without any influences on the current relationships between them and schools. They could also skip questions that they did not want to answer. We did not collect any individual data for confidentiality; hence, re-identifying students’ identifications is impossible. We also offered the hotline in the package if students needed helps. Both students and their parents/guardians signed into the written informed consent. The institutional review board of the Hanoi Medical University granted the study protocol (Code: 22NCS17/HDDDDHYHN)..Among 712 secondary school students participating in the study, there were 414 female students and 298 male students. The 3-month prevalence of physical, social aggression, verbal, and sexual bullying experiences were 8.4%, 31.2%, 11.9%, and 2.7%, respectively. The 3-month prevalence of being physically bullied among children aged 12 and 13 years old was two times higher than that in those aged 11 and 14 (p<0.05). The 3-month prevalence of emotional bullying experience was different among age groups and body mass index groups (p<0.05). Male students (4.4%) had approximately three times higher in the rate of sexual harassment compared to female counterparts (1.5%) (p<0.05) . Meanwhile, the most common behaviour of social aggression was “Someone disrespects and refuses to listen to my opinion” (24.0%), following by “Takes control and makes me do what they want” (10.0%). “Talks profanely looks down on me, my family, makes fun of, mimics or imitates my behaviour” was the most frequent verbal bullying act that students suffered from (7.7%), following “Discusses, gossip, or spread bad rumours about me” (6.5%). Finally, “Touches a sensitive part of my body” (2.1%) and “Pulls, takes off, tugs skirt/shirt/pants” (1.5%) were the two most common acts of sexual bullying that the students experienced in the last three months. Overall, 24.4% of students suffered from one type of bullying, 9.7% suffered from two types, and 3.4% experienced three types or more.Slapping, hitting, kicking, or beating were the most common behaviour of physically bullying (8.2%) Table 3 indicates that students who had physical bullying, social aggression, and verbal bullying experience perceived significantly lower levels of support from family, classmates, and teachers and school security compared to non-victims (p<0.05). For sexually bullying, only levels of support from friends were differently perceived between those with and without experience (p<0.05).Table 4. Students receiving higher levels of support from classmates  and family  had a lower likelihood of being physically bullied. These associations were also found in verbal bullying experience when a higher level of support from classmates  and family  were negatively associated with being verbally bullied. Having a higher level of support from family was related to a lower odd of having social aggression experience. Meanwhile, students who were overweight or obese were more likely to suffer social aggression  compared to those with normal BMI. Females had a lower likelihood of having sexually bullying experience compared to male students. The sexual bullying experience was also lower in students aged 13 and 14 years old compared to those aged 11 years old.The results of the multivariate analysis are shown in Table 5 shows that students having bullying experience had lower EQ-5D index, higher rates of depression, anxiety, and stress compared to those without experience (p<0.05), except for the anxiety rate among those with and without sexual bullying experience.When examining the difference of HRQOL, depression, anxiety, and stress between those with and without bullying experience, Table 6 depicts the associations between bullying experience, HRQOL, and different mental problems. After adjusting to other covariates, students suffering from physical bullying , social aggression  and verbal bullying  had significantly lower HRQOL compared to non-victims. All of the bullying forms were found to be associated with higher odds of stress. Students experiencing social aggression and sexual bullying had a higher likelihood of developing depression. Meanwhile, students experiencing social aggression were more likely to suffer anxiety compared to those without experience .Our findings showed that the most common form of bullying among adolescents in urban settings in Vietnam was social aggression, following by verbal bullying, physical bullying, and sexual bullying. This study also showed negative relationships between being bullied and adolescents’ quality of life and mental health, as well as the active role of family-friends-school support in bullying prevention.Our study echoed the previous findings that bullying experience was a widespread phenomenon among adolescents in the metropolitan area of Vietnam , 31. In Social aggression was the most common type of bullying among students in urban secondary schools, following by physical, verbal, and sexual bullying. This result was different in findings from studies in other countries such as India, where “teasing, making fun, taking things, and making feels sad” were the most common bullying acts , 35. IndOur multivariate results reaffirmed the negative relation between age and a likelihood of being bullied. During the transition time from primary to secondary school (e.g. sixth and seventh grades), students may be more likely to find social dominance to prove their power over their peers, which might put them at a high risk of being bullied, especially physical and sexual bullying , 38. NotOverweight and obesity students were more likely to experience social aggression. In prior literature, overweight and obese youths and adolescents were also found to be associated with bullying victimization . In thisOur findings were in line with the previous studies to show that positive parent-child, friend-friend, and teacher-student relationships were the significant protective factors from being bullied –46. PrevThe findings of this study contributed to the current literature about the effects of different types of bullying on students’ HRQOL and mental health. Prior studies showed that having bullying victimization experience increased significantly the risk of depression as well as suicidal ideation , 49, 50.Our study has some limitations. Firstly, although the study used a multi-stage sampling method and involved a simple random sampling approach to select study participants, our results may not reflect fully the bullying phenomenon among adolescents in urban Viet Nam. In addition, our study did not include adolescents who were out-of-school, not at school during the study period, or in private schools. Second, our study used a cross-sectional design, which was unable to develop causal relationships between bullying and associated factors, as well as relationships with mental health status and quality of life. Third, we did not investigate the prevalence of bullying perpetrators among our sample given sensitive information. Moreover, this study did not investigate the positive and negative influence of social media, as well as accessibility of different programs in preventing school bullying in the community, which might be important to predict the bullying experience in the adolescents. Finally, our study asked participants about the bullying experiences over the past 3 months, which may lead to recall bias as well as miss any events occurring later than 3 months. We did not used a validated instrument to measure bullying experience, which might reduce our comparability to other studies. Further studies with holistic approaches should be warranted.This study indicated a high 3-month prevalence of bullying experience, especially social aggression, among students in metropolitan secondary schools in Vietnam, as well as the decrement of HRQOL and high risks of mental problems in bullying sufferers. Holistic approaches involving family, peers, and schools, along with enhancing school security, are potential methods to reduce the impact of bullying on adolescents’ life and well-being.


Cage rearing has critical implications for the laying duck industry because it is convenient for feeding and management. However, caging stress is a type of chronic stress that induces maladaptation. Environmental stress responses have been extensively studied, but no detailed information is available about the comprehensive changes in plasma metabolites at different stages of caging stress in ducks. We designed this experiment to analyze the effects of caging stress on performance parameters and oxidative stress indexes in ducks.Liquid chromatography tandem mass spectrometry (LC/MS-MS) was used to determine the changes in metabolites in duck plasma at 5 (CR5), 10 (CR10), and 15 (CR15) days after cage rearing and traditional breeding (TB). The associated pathways of differentially altered metabolites were analyzed using Kyoto encyclopedia of genes and genomes (KEGG) database.The results of this study indicate that caging stress decreased performance parameters, and the plasma total superoxide dismutase levels were increased in the CR10 group compared with the other groups. In addition, 1,431 metabolites were detected. Compared with the TB group, 134, 381, and 190 differentially produced metabolites were identified in the CR5, CR10, and CR15 groups, respectively. The results of principal component analysis (PCA) show that the selected components sufficiently distinguish the TB group and CR10 group. KEGG analysis results revealed that the differentially altered metabolites in duck plasma from the CR5 and TB groups were mainly associated with ovarian steroidogenesis, biosynthesis of unsaturated fatty acids, and phenylalanine metabolism.In this study, the production performance, blood indexes, number of metabolites and PCA were compared to determine effect of the caging stress stage on ducks. We inferred from the experimental results that caging-stressed ducks were in the sensitive phase in the first 5 days after caging, caging for approximately 10 days was an important transition phase, and then the duck continually adapted. Ducks are the most widely domesticated waterfowl, and are mainly bred in China ,2. With Chronic stress in animals is divided into two stages, the sensitive phase and the adaptable phase. As a major abiotic stress that negatively affects duck performance, caging stress is a nonpathological chronic stress, and tolerance to caging stress strongly depends on the stress response abilities of ducks. Physiological adaptations can be manifest at many levels, and activation of the hypothalamic–pituitary–adrenal (HPA) axis induces many biological mechanisms to re-establish homeostatic conditions and maintain physiological activity ,8. OtherMetabolites in the blood participate in the stress response and related functions by activating or deactivating related pathways. Metabolomics has been used to systematically identify changes in metabolites in organisms and elucidate their responses to environmental changes, growth or various diseases. Metabolomics has provided new perspectives to investigate the evolution of stress responses, and mass spectrometry is a good approach for discovering new biomarkers. Stress studies have identified a variety of metabolites associated with food intake, egg production, and body weight, but those metabolites that play physiological functions in the transition from the stress sensitive phase to the adaptable phase have received less attention.As a new breeding model, few reports have examined biomarkers of cage-rearing stress in ducks. Based on this background, the physiological conditions of the caging-stress response of ducks were investigated by measuring production performance and plasma biochemical indexes after the ducks were placed in cages. For this experiment, we used untargeted metabolomics based on liquid chromatography-mass spectrometry (LC-MS) to investigate metabolite perturbations and to identify associated metabolic pathways and plasma biomarkers. The purposes of this study were to provide valuable biochemical information, to determine the mechanisms of chronic stress responses in cage-reared ducks, and to define biomarkers to assess cage-rearing stress in ducks.The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to and that appropriate ethical review committee approval has been received. The methods were conducted according to the Guidelines for Experimental Animals established by the Ministry of Science and Technology. All procedures involving animal subjects were approved by the Animal Ethics Committee of Hubei Academy of Agricultural Sciences. The authors confirm that they have followed EU standards for the protection of animals used for scientific purposes. We obtained written informed consent to use the animals in our study from the owner of the animals.Anas platyrhynchos, Lihu Duck Farm, Hubei Province) weighing 1,280 to 1,620 g were used. The experiment was designed to include random assignment to 2 processes, namely, cage-rearing treatment and floor rearing, and 5 repetitions (n = 6). In the trial group, the ducks were raised in a stepped duck coop, which was welded together by steel wire. The dimensions of the cages were 40 cm long, 60 cm wide and 50 cm high, and 2 ducks were placed in one cage. The ducks were raised under a light cycle of 18 hours of light and 6 hours of darkness. All ducks were fed the same diet, which met the requirements. The amount of feed provided to each bird was 160 g/d. An automatic water supply system provided clean water for the birds.Sixty female 120-day-old mallards  using an electronic balance.Samples of fasting venous blood were collected from the wing veins of ducks in the following 4 stages: traditional breeding (TB), 5 days after cage rearing (CR5), 10 days after cage rearing (CR10), and 15 days after cage rearing (CR15). Then, at various time points, the selected ducks were immediately anesthetized with sodium pentobarbital  and euthanized by exsanguination. The blood sample was collected in a tube containing anticoagulant and centrifuged at 3,000×g and 4°C for 10 min to obtain the plasma, which was flash frozen in liquid nitrogen, and all samples were quickly stored at −80°C until further processing.The plasma levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and malondialdehyde (MDA) were determined with clinical chemistry assay kits according to the manufacturer’s instructions . The SOD, MDA, and GSH-PX contents were determined at 550 nm, 532 nm, and 412 nm, respectively, by performing a quantitative colorimetric analysis. We calculated the concentrations of these substances based on the optical density and the standard curve according to the formula provided in the instructions.The plasma samples (100 μL) were sent to Novogene  for an untargeted metabolomics analysis. The samples were individually resuspended in prechilled methanol and 0.1% formic acid by vortexing well. The samples were incubated on ice for 5 min and then centrifuged at 15,000 rpm at 4°C for 5 min. A portion of the supernatant was diluted to a final concentration of 60% methanol with LC-MS grade water. The samples were subsequently transferred to a fresh Eppendorf tube with a 0.22 μm filter and then centrifuged at 15,000×g for 10 min at 4°C. Finally, the filtrate was injected into the liquid chromatography tandem mass spectrometry (LC-MS/MS) system for analysis.Equal volumes of each experimental sample were mixed and marked as quality control (QC) samples. The blank sample was the blank matrix of the experimental sample, and the pretreatment process for the blank sample was the same as that for the experimental samples.LC-/MS-MS analyses were performed using a Vanquish UHPLC system  coupled to an Orbitrap Q Exactive HF-X mass spectrometer . Samples were injected onto a Hyperil Gold column  using a 16 min linear gradient at a flow rate of 0.2 mL/min. The eluents for positive polarity mode were eluent A (0.1% formic acid in water) and eluent B (methanol). The eluents for negative polarity mode were eluent A  and eluent B (methanol). The solvent gradient was set as follows: 2% B, 1.5 min; 2% to 100% B, 12.0 min; 100% B, 14.0 min; 100% to 2% B, 14.1 min; and 2% B, 16 min. A Q Exactive HF-X mass spectrometer was operated in positive/negative polarity mode with a spray voltage of 3.2 kV, a capillary temperature of 320°C, a sheath gas flow rate of 35 arb and an aux gas flow rate of 10 arb.https://www.mzcloud.org/) and ChemSpider (http://www.chemspider.com/) databases to obtain the accurate qualitative and relative quantitative results. The significance of the difference between groups was analyzed according to metabolite expression levels. Principal component analysis (PCA), partial least squares-discriminate analysis (PLS-DA), variable importance in projection (VIP ≥1.0), and fold change analysis  were performed as described in a previous report. Then, Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses (http://www.genome.jp/kegg/) of differentially expressed compounds were performed. The most enriched pathway terms with p-values <0.05 in the CR vs TB comparison were selected.The raw data files generated by ultra-high performance liquid chromatography–tandem mass-spectrometry (UHPLC-MS/MS) were processed using Compound Discoverer (CD) 3.0 software to perform peak alignment, peak picking, and quantitation for each metabolite. Afterwards, the peak intensities were normalized to the total spectral intensity. The normalized data were used to predict the molecular formula based on additive ions, molecular ion peaks and fragment ions. Then peaks were matched with the mzCloud , biosynthesis of unsaturated fatty acids (arachidonic acid), and phenylalanine metabolism  . The difModern production management and breeding technology may lead to stress reactions. As a new breeding model, cage rearing of ducks increases productivity and serves as a tractable model for studying adaptation to stress in animals. The production performance of livestock and poultry is altered during the initial response to stress. Our research shows that caged rearing decreased the duck body weight and food intake in the first 5 days and gradually increased in the subsequent 10 days, with statistically significant differences. Lei et al  reportedThe investigation of changes in blood indexes of caging-stressed ducks provides a theoretical basis for revealing the different stages of duck caging stress. No obvious changes in the levels of MDA and GSH-PX in plasma were observed after caging, the plasma T-SOD levels increased significantly after 10 days. Changes in organisms during stress are postulated to be linked to accumulating lipid peroxidation products, such as free radicals, peroxy radicals, hydroperoxides, aldehydes, and ketones . AntioxiWhen stress occurs, animals have been shown to activate predicable, adaptive responses to stress in many systems, including the metabolic, cardiovascular, neuroendocrine, and even digestive systems . Plasma Based on acquired differentially abundant metabolites, PCA was performed to investigate the presence of intrinsic clusters within the data matrix. The PCA showed a striking pattern in plasma samples clustered according to the experimental timepoint, with a progressive shift from the TB group through the CR5, CR10, and CR15 groups. An obvious distinction between the TB groups and CR10 groups in positive mode and negative mode was observed, representing the inherent metabolic difference between these two groups. The PCA revealed the clustering of samples, which can be used as a method to identify different states and has been showed in many studies that applied metabolomics methods. According to the PCA results from the study by Cui et al  LC-MS/MSA KEGG pathway enrichment analysis was carried out on the acquired differentially abundant metabolites. Several pathways or biological processes were identified and may provide insights into the underlying response mechanism of caging stress. Ovarian steroidogenesis, and biosynthesis of unsaturated fatty acids were significantly different between ducks reared without cages and ducks in the CR5 group. L-Phenylalanine, N-acetyl-L-phenylalanine, platelet-activating factor (PAF), arachidonate, oleic acid, progesterone and cortisol are the key metabolic compounds in the aforementioned metabolic pathways. Significant changes in intermediates involved in the PTS, aldosterone synthesis and secretion, vitamin digestion and absorption, and steroid hormone biosynthesis were observed in ducks from the CR10 group ducks compared with the TB groups. Arachidonate, corticosterone, progesterone, ascorbate vitamin C (VC), retinoic acid, and VB2 are key metabolic compounds involved in these metabolic processes. Ovarian steroidogenesis, arachidonic acid metabolism, and regulation of lipolysis in adipocytes were significantly different between ducks reared without cages and ducks in the CR15 group. Arachidonate, leukotriene B4, progesterone, corticosterone, cortisol, DHA, and EPA are key metabolic compounds involved in the aforementioned metabolic processes. The metabolites detected in this study showed that, the differential metabolites in the caging-stress groups were strongly associated with steroid hormone biosynthesis and regulation, lipid metabolism and oxidation-reduction reaction signaling pathways. Steroid hormones participate in organ development, reproduction, body homeostasis, and stress responses . BecauseN-acetyl-L-phenylalanine, L-phenylalanine, PAF, and oleic acid showed significant differences in metabolite ratios between the CR5 and TB groups. Aromatic amino acids, particularly L-phenylalanine, are known to increase plasma cholecystokinin levels and reduce food intake in humans . The abnThe metabolic ratios of VC, riboflavin (VB2), and retinoic acid were significantly different between the CR10 and TB groups, and these metabolites participate in the process of oxidative stress and lipid peroxidation. The VC is an antioxidant because it prevents other compounds from being oxidized . The VC Significant differences in the ratios of the metabolites leukotriene B4, DHA, and EPA were observed between the CR15 and TB groups, and these metabolites play major roles in the anti-inflammatory process and in regulating the viscosity of cell membranes . These fIn summary, in this study, the production performance, blood indexes, number of metabolites and PCA were compared to determine the caging stress stage of ducks. We inferred from the experimental results described above that caging-stressed ducks were in the sensitive phase in the first 5 days post after caging, caging for approximately 10 days was an important transition phase, and then the duck continually adapted. Metabolic signatures were presented and biomarkers were defined to evaluate the stress status of caged ducks. This research can serve as the basis for further investigations of duck caging stress and provides a new perspective for studying the mechanism of animal stress.


Acute rectal ischemia is a rare entity because the rectum has abundant blood supply from the inferior mesenteric, internal iliac, internal pudendal, and marginal artery with rich collaterals.We present a case of an acute ischemic proctosigmoiditis with a history of rectal cancer who completely recovered after total neoadjuvant treatment and was in the “watch-and-wait” protocol. Urgent laparoscopic low anterior resection and protective ileostomy were performed. Causes of acute rectosigmoid ischemia include old age, diabetes, atherosclerosis, previous aortic surgery due to aneurysm, vasculitis, and radiotherapy. Ischemia may be present as only involving the mucosa, which can be managed conservatively, but full-thickness necrosis requires urgent surgical intervention. Endoscopic examination is the gold standard in initial diagnosis. Ischemic gangrene of the rectosigmoid colon is a rare condition and can be life-threatening unless an urgent surgical intervention is performed. Acute ischemia of the rectosigmoid colon is an extremely rare condition due to rich collateral blood network and can vary between 2% and 5% of all cases of acute ischemic colitis -3. IscheHerein, we present the case of a male patient who completely recovered from local advanced rectal cancer (cT3N positive) following total neoadjuvant treatment (TNT)  and was 2 twice daily during radiotherapy. After four weeks, TNT was administered, and the consolidation chemotherapy regimen was oxaliplatin 130 mg/m2 on day 1 plus capecitabine 1,000 mg/m2 twice daily on days 1-14 every three weeks for eight cycles. In the patient, the time between cCR and developing of acute ischemia was 14 months.A 58-year-old male patient was admitted to our clinic with complaints of abdominal pain, fever, weakness, and bloody stool. An initial assessment revealed the following vital parameters: blood pressure of 110/70 mmHg, pulse of 80 beats/minute, respiratory rate of 20 breaths/minute, saturation of 97% on room air, and temperature of 38°C. The patient had a local advanced rectal cancer history, which achieved a complete clinical response (cCR) after TNT, and he was one of our patients in the WW program  count of 14,000/μL, neutrophil count of 7,240/ μL, and C-reactive protein level of 23 mg/ μL. Abdominal computed tomography (CT) revealed concentric and irregular thickening of the rectosigmoid colon with stranding of the adjacent fat from up to the mid-rectum and distal sigmoid colon, and signs of air bubbles in the colonic wall Figure . IntraveIschemic colitis is the most common form of gastrointestinal ischemia, accounting for 50% to 60% of all cases and occurring with an incidence of 4.5 to 44 cases per 100,000 person-years .The underlying mechanism leading to ischemic proctosigmoiditis is not completely understood.Bharucha et al.  reviewedThe clinical presentation of patients with ischemic proctosigmoiditis may be similar to those with inflammatory bowel disease or pseudomembranous colitis and lead to misdiagnosis . HistoloHerein, we reported acute ischemic proctosigmoiditis in rectal cancer patients in the WW protocol. To the best of our knowledge, this is the first ever reported case among the WW protocol patients. The patient was in our close follow-up, and his two months earlier endoscopic examination was normal. The cause of ischemia in our patient is unclear. We conjecture that previous radiotherapy may cause this condition. Since the distal part of the rectal mucosa appearance seemed healthy, we performed laparoscopic LAR and protective ileostomy, with the latter closure being performed four weeks later. Our patient is feeling healthy and his quality of life is pretty good.Ischemic gangrene of the rectosigmoid colon is a rare emergency condition. Ischemic gangrene of the rectum may be rarely seen in patients with rectal cancers after radiotherapy. Endoscopic and CT examination is necessary for diagnosis and treatment options. Immediate surgical intervention is inevitable in full-thickness ischemia of the rectosigmoid colon.


In the near future, COVID-19 vaccine efficacy trials in larger cohorts may offer the possibility to implement child and adolescent vaccination. The opening of the vaccination for these strata may play a key role in order to limit virus circulation, infection spreading towards the most vulnerable subjects, and plan safe school reopening. Vaccine hesitancy (VH) could limit the ability to reach the coverage threshold required to ensure herd immunity. The aim of this study was to investigate the prevalence and determinants of VH among parents/guardians toward a potentially available COVID-19 vaccination for children and adolescents. An online survey was performed in parents/guardians of children aged <18 years old, living in Bologna. Overall, 5054 questionnaires were collected. A vast majority (60.4%) of the parents/guardians were inclined to vaccinate, while 29.6% were still considering the opportunity, and 9.9% were hesitant. Highest vaccine hesitancy rates were detected in female parents/guardians of children aged 6–10 years, ≤29 years old, with low educational level, relying on information found in the web/social media, and disliking mandatory vaccination policies. Although preliminary, these data could help in designing target strategies to implement adherence to a vaccination campaign, with special regard to web-based information. Since March 2020, most governments around the world have closed schools and other educational institutions in an attempt to contain the diffusion of the COVID-19 pandemic, with an estimated dramatic impact on the education of over 850 million children and youths ,4. On thIn Italy, school activities were suspended on 4 March 2020, based on the epidemiological evolution and the progressive viral spread on the national territory. The initial suspension turned into a protracted closure due to the subsequent lockdown of the whole country , and theIn the Emilia-Romagna Region (ERR), positive cases of SARS-CoV-2 within the school community have been tracked since the resumption of educational service activities through a dedicated screening system operated by the Local Health Authority, with the aim of identifying and managing any possible outbreak within classes and schools. Specifically, the entire class is put into quarantine every time in case of outbreak (>1 case), or when a single positive case occurs in a community with a high risk of COVID-19 transmission  is not mandatory] . These mIn December 2020, the European Centre for Disease Prevention and Control (ECDC), based on the data on the experience of school closure in European countries from August to December 2020, recommended to consider this measure as a last resort in the pandemic management . AccordiAt the end of December 2020, the vaccination campaign against SARS-CoV-2 began in Italy, initially involving healthcare professionals and residents of Long-Term Care Facilities (LTCFs) considered at high risk of exposure and severe outcomes, respectively. The vaccination schedule intended to involve teachers and school staff as early as the second phase of the vaccination campaign . ChildreThe aim of this study was to investigate the prevalence and determinants of VH among parents/guardians toward COVID-19 vaccination in children and adolescents, so as to orient future awareness-raising tools and vaccination strategies.An online survey was performed in parents/guardians of children aged <18 years old, living in the Metropolitan City of Bologna, recruited between December 2020 and January 2021 among family members interviewed by the personnel of the local Public Health Service dedicated to school-based SARS-CoV-2 screening. Recruitment was obtained through emails sent to principals, who were in charge of dissemination. Adhesion to the study was voluntary; data were collected anonymously. All participants provided online informed consent to be included in the database for study participation.Socio-demographic variables of respondents and their children were collected. Participants were also asked whether their children had received mandatory vaccinations . All analyses were carried out using Stata Statistical Software 15 .Variables were described as absolute frequencies and percentages. Determinants of VH were assessed by uni- and multivariate analyses. Results of multivariate analyses are presented as an odds ratio (OR) with standard error (SE) and a 95% confidence interval (95% CI). A backward stepwise analysis was run to define the variables to be included in the final multiple logistic regression model, according to the results of univariate models and to the principles of parsimony and biological plausibility. Statistical significance was set at Overall, 5054 questionnaires were collected. Fifty-four had to be excluded as participants denied the consent to the treatment of personal data, and seven for non-compliant data (children’s age and school grade were not consistent), leaving 4993 questionnaires available for data collection and analysis. The main parent/guardian demographic and education data are reported in N = 3824) were females; the great majority  were aged between 40 and 49 years old, while few  were younger than 30. With regard to the level of education, most  had a high-school qualification, followed by those with a university degree , a post-university Master’s degree , and finally, those with a primary school qualification  , ≥14  and, finally, 0–5 years   a. Analys; 12.7%) b.The exploration of adherence to Italian obligatory vaccination law resulted in 29 (0.6%) non-adherent, 118 (2.4%) partially adherent, and 4846 (91.1%) fully adherent.When exploring the inclination to have their children receive a COVID-19 vaccination, the results show that 496 (9.9%) did not intend to vaccinate their children, 1480 (29.6%) did not know, and 3017 (60.4%) were inclined to have their children receive a COVID-19 vaccination.Sources of information chosen by parents/guardians to decide regarding COVID-19 vaccination in children are reported in p < 0.05) , as well as attitudes toward mandatory vaccination policy before and after the COVID-19 pandemic significantly correlated to VH ( < 0.05) . Specifin = 358, 72.6%) had a lower level of education, whereas those who had completed postgraduate studies had a 90% level of reliance on physicians (n = 514). Moreover, 6% (n = 34) of the most educated people used the Web and/or Social Media as a principal source of information to decide about vaccination  of the participants agreed with the Law n.119/2017 [n = 469) were undecided, while 6.1% (n = 307) disagreed—strongly in 2.2% (n = 108) of the cases  or strongly disagreed   b.We report the results of a survey performed in a large cohort of parents/guardians assessing the rate of vaccine hesitancy (VH) toward potential COVID-19 vaccination in their children aged 0–18 years old, and its determinants.Our study, including 5054 parents/guardians of the Metropolitan city of Bologna in the north of Italy, observed a 40% rate of VH, significantly higher if compared to the general Italian population 22.7–29.1%) or university students (13.9%) ,20,21,22.7–29.1% Worldwide mass vaccination is envisaged as key strategy to mitigate the effects of the COVID-19 pandemic . TherefoThree types of COVID-19 vaccines  are currently being tested in children . MeanwhiOnly a few studies have assessed parent/guardian VH toward COVID-19 vaccination in the pediatric population, showing extremely heterogeneous results (i.e., 72.6% reported by a Chinese study  vs. 75.8A possible explanation for the higher overall rate of VH demonstrated by our study could be the perception of a low risk of severe COVID-19 disease in children, especially by younger parents/guardians, which confirmed results of previous studies regarding the higher confidence in and propensity toward vaccination of their children of parents/guardians aged ≥50 ,33. In aIn line with other studies, females were more hesitant than males ,33,34. TParticularly interesting are the media-related data from our study: The source of information significantly influenced attitudes toward COVID-19 vaccine, and varied with educational levels. In particular, 84.4% of parents/guardians mainly relied on medical advice; personal beliefs, web/social media, and television; the opinion of religious authorities/politicians/celebrities were not taken into account by most, although people with lower levels of education were less likely to consult physicians in favor of the Web and social media. These factors may suggest that a higher level of education provides a protective effect against VH by giving more tools for decision-making, without falling prey to conspiracy theories. Moreover, as reported by other groups, those who mainly relied on Web sources and/or social media tended to be more hesitant ,41,42,43Despite a heavy impact on school activities of policies against COVID-19, it seems that VH and disagreement with the so-called “Lorenzin” Law have increased. It would be important to investigate this increase with further studies and specifically to evaluate the effects of school policies on VH.The main strengths of the study are the large cohort of enrolled subjects, the homogeneity of the geographic origin, the absence of operator influence on questionnaire-filling, and the homogeneous distribution of both children and parents/guardians for age, gender, and level of education . On the In conclusion, parents’ or guardians’ VH toward the COVID-19 vaccine in their children was observed to be higher than for other vaccines; female gender, younger parental/children’s age, lower educational level, and referral to the Web/social media were found to significantly increase the risk of VH. Although preliminary, these data could help in designing target strategies to implement adherence to vaccination campaigns, with special regard to Web-based information.


Mast cells are highly differentiated, widely distributed cells of the innate immune system, that are currently considered as key regulators of both innate and adaptive immunity. Mast cells play a key role in health and survival mechanisms, especially as sentinel cells that can stimulate protective immune responses. On the other hand, it has been shown that mast cells are involved in the pathogenesis of several diseases, and recently a possible pathogenetic role of mast cells in diabetes has been proposed. In this review we summarize the evidence on the increased presence of mast cells in the pancreas of subjects with type 1 diabetes, which is due to the autoimmune destruction of insulin secreting beta cells, and discuss the differences with type 2 diabetes, the other major form of diabetes. In addition, we describe some of the pathophysiological mechanisms through which mast cells might exert their actions, which could be targeted to potentially protect the beta cells in autoimmune diabetes. Mast cells  were fir+/CD117+ haemopoietic stem cells in the bone marrow [Mast cells are terminally differentiated, highly distributed, granular cells of the immune system originating from CD34e marrow ,6. Recene marrow . This wie marrow .TC containing both tryptase and chymase, and MCT expressing only tryptase [In both rodents and humans, mast cells appear to be heterogeneous in nature ,10,11. DMast cells express an array of activating and inhibitory receptors (mast cells have been claimed to express more receptors than any other cell type) on their surface, which enables these cells to recognize and respond to a wide spectrum of both exogenous pathogens and endogenous molecules derived from damaged or inflamed tissues ,13. Mastde novo synthesized lipid metabolites of arachidonic acid, such as prostaglandins and leukotriens. The third group includes various cytokines and chemokines which are synthesized in response to stimulation through unregulated gene expression.When activated, mast cells release a large variety of mediators which have been classified on the basis of the kinetics of their release ,20. The In view of the large amount of secreted mediators (no other cell is thought to make more mediators), performing a variety of different biological functions, it is not surprising that mast cells are currently considered not simply as effector immune cells, but rather as key regulators of both innate and adaptive immunity ,22. It hThus, mast cells can be considered key players in health and survival mechanisms, especially as sentinel cells that sense pathogens and stimulate protective immune responses. Indeed, there are no humans without them. On the other hand, mast cells are involved in the pathogenesis of many diseases . In factHowever, phylogenetic studies showing that mast cells can be found even in animals lacking immunoglobulins, together with the variety of mediators released upon mast cell activation, suggest that these cells could be involved in the pathogenesis of several diseases besides those requiring IgE . In partHowever, in other circumstances, mast cells can contribute to the restoration of homeostasis. In mammals, a positive role of mast cells in inflammation has been identified by using mast-cell-deficient mice as experimental models ,40. OtheTherefore, mast cells are sophisticated cells of the innate immune systems, which are able to carry out a wide spectrum of pleiotropic effects, either deleterious or potentially protective.(1)Type 1 diabetes, in most patients caused by autoimmune destruction of pancreatic beta cells (type 1A) and in some cases of non-autoimmune, unknown origin ;(2)Type 2 diabetes, caused by variable degrees of beta cell functional mass loss, often in the background of reduced insulin sensitivity;(3)Specific types of diabetes, due to several different causes;(4)Gestational diabetes.Diabetes mellitus is a heterogenous disorder of the metabolism of carbohydrate, fat and protein, due to the interplay of genetic and environmental factors . It is cIn type 1 diabetes, which represents up to 10% of all cases, the key pathophysiological feature is the autoimmune destruction of beta cells, mediated by the interaction of innate and adaptive immunity ,46,47. IIt has been estimated that the global prevalence of diabetes in 2019 (age 20–79 years) was >9% (463 million people), which is expected to increase to 10.2% by 2030 and 10.9% by 2045 . In addiMorbidity and mortality in diabetic patients are high ,48,54. CIn summary, diabetes is one of the most prevalent non-communicable diseases, carrying an ominous clinical and economic burden. Although heterogenous in its manifestations and mechanisms, diabetes occurs and progresses when pancreatic beta cells fail to produce sufficient amounts of insulin to regulate blood glucose levels and other metabolic processes. Much attention is being therefore pointed to the pathophysiology of insulin-secreting cells in the pancreas.2 (mean ± SEM), whereas in those with mild amyloidosis (<2% of the islet area) the number of mast cells within the islets was 31.6 ± 3.3 per mm2. In diabetic individuals there was a significantly increased degree of islet amyloidosis and an approximately 3-fold larger number of intra-islet mast cells (61.5 ± 19.5 per mm2), which correlated with the extent of islet amyloidosis. Shortly afterwards, it was shown that the increase of mast cells in the pancreatic islets can occur independently from amyloidosis and appears therefore to be a direct consequence of diabetes [Initial evidence of the presence of mast cells in the pancreas dates back to the late 1950s , but mordiabetes . Interesdiabetes . In compdiabetes . Successdiabetes ,60,61. I2 in pancreatic samples from non-diabetic and type 1 diabetic donors, respectively (p  <  0.05) However, the number of mast cells in specimens from donors with type 2 diabetes (6.6  ±  1.3 per mm2) was similar to that of controls. In further detail, the number of mast cells per islet was more than 3-fold higher in type 1 diabetes  than in control samples. Accordingly, the percentage of islets with at least one mast cell was 94% in type 1 diabetic and 27% in non-diabetic individuals (p < 0.05). Notably, several mast cells in the samples from type 1 diabetic islets looked partly degranulated .Only recently, however, the role of mast cells in the pancreas of diabetic individuals has been investigated. Martino and colleagues  examinedanulated , indicatAs for the other immune system cells examined, and in comparison with non-diabetic samples, lymphocyte number in pancreatic tissues was significantly greater in type 1 diabetes, and macrophage number was significantly greater in type 2 diabetes .2 tissue were 1.4 ± 0.60 in controls, 2.3 ± 0.49 type 1 diabetic donors (p = 0.062 vs. controls), and 2.4 ± 0.57 in autoantibody-positive individuals (p = 0.068 versus controls). However, significant differences were observed in the amount of mast cell subtypes. The proportion of tryptase-positive mast cells was >90% of total mast cells in type 1 diabetic donors, and about 50% in autoantibody-positive and control groups. Interestingly, the number of tryptase-positive cells was significantly greater in type 1 diabetic donors  and auto-antibody-positive subjects  than in non-diabetic control individuals (0.81 ± 0.57). However, and in line with the results of a previous study [More recently, pancreas samples were obtained from 47 organ donors ) . Electrous study .Therefore, the number of mast cells and/or their certain subtypes is increased in the pancreas and the islets of subjects with type 1 diabetes, but not in those of people with type 2 diabetes, suggesting a role of mast cells in autoimmune diabetes.p = 0.045), with 25% of the animals remaining diabetes-free until the end of the study (130 days). Overall, disodium cromoglycate therapy reduced the risk of disease by about one-half, and the animals without diabetes at 130 days had normal islet histology.Some evidence supports the involvement of mast cells in the pathophysiology of type 1 diabetes. In the BioBreeding (BB) rat, the most extensively studied rat model of type 1 diabetes, it was found that the abundance of mast cells in peripheral lymph nodes is significantly increased and that eotaxin, a chemokine that can recruit mast cells, is expressed in the pancreatic islets, being specifically localized in the beta cells ,65. Of i(W-sh/W-sh)), it was observed that both incidence and progression of diabetes in this model were independent of mast cells [A role of mast cells in the pathogenesis of type 1 diabetes has been proposed by some authors in the type 1 diabetes NOD mouse model . In thesst cells . In the st cells . At the st cells .Mast-cell-deficient mice developed severe insulitis and rapid progression of hyperglycemia, with 100% of mice becoming diabetic. This was associated with reduced number of T regulatory (Treg) cells in the pancreatic lymph nodes. Furthermore, there was marked reduction of IL-10, TGF-β, and IL-6 in the pancreatic tissue. Of note, mast cell adoptive transfer in this model before streptozotocin-injection-induced resistance to diabetes development, which was associated with increased Treg cells and decreased IL-17-producing T cells .p < 0.01). In addition, the percentage of islet insulin-positive cells showing TUNEL-positive nuclei was significantly greater after incubation with the amine than in control islets (2.8 ± 1.6% vs. 0.6 ± 0.5%). Intriguingly, activation of caspases 9 and 3, key factors in apoptotic processes, was not affected. However, it was found that the expression of apoptosis-inducing factor  was significantly increased after exposure to histamine [A few sets of data have been generated also with human islets ex vivo. Histamine is one of the most abundant secretory products of mast cells, and its role was tested by direct exposure of isolated human islets to this compound for 72 h . It was istamine . AIF is istamine . The impistamine .These discrepant results may be due to the different experimental designs used and/or to the possibility that mast cells might have species specific deleterious or protective actions.Mast cells are highly differentiated, widely distributed cells of the innate immune system. The involvement of mast cells in diabetes is corroborated by findings indicating that these cells are associated with inflamed adipose tissue, the development of certain diabetes complications such as diabetic nephropathy, and reduced wound healing in the case of diabetic foot lesions ,71,72,73