The shortage of health care workforce is a common problem all over the world and one of the main reasons for the shortage is the high turnover rate. Based on the characteristics of medical work, this study explored the relationship among public service motivation (PSM), work stress, task performance and turnover intention.Medical personnel in public hospitals were selected by stratified random sampling in Jilin province of China and validated scales from previous studies were applied to measure the variables. Besides, a structural equation model of turnover intention was constructed to demonstrate the relationship.β = −0.292, P < 0.001), work stress had direct and positive effects on the turnover intention , whereas task performance had no significant effect on turnover intention . The results showed an acceptable fit model.A total of 3191 valid questionnaires were collected. The results showed that the score of turnover intention was 2.02 ± 1.13. There are significant differences in turnover intention among medical staff of different genders and departments. At the same time, PSM had direct and negative effects on the turnover intention (The greater the PSM, the lower the turnover intention, and the higher the work stress, the higher the turnover intention. In addition, work stress and task performance play a mediating role between PSM and turnover intention. This paper provides theoretical support for the measures to reduce the turnover intention of medical staff. Reports from the Global Health Workforce Alliance (GHWA) and World Health Organization (WHO) show that it is difficult for health posts to attract and retain a heath care workforce, so health care workforces are lacking worldwide . The shoChina does not lack a “reserve” medical staff, but it cannot effectively transform and retain medical staff. Between 2005 and 2014, China produced 4727,977 clinical medicine graduates but only registered 75,223 clinicians . This paAccording to the theory of behavioral planning, behavioral intention can predict actual behavior well , and sinScholars generally accept that turnover intention is an antecedent variable and the final state of withdrawal cognition before actual turnover. The antecedents of turnover intention can be divided into macrocosm and microcosm factors. Macrocosm factors include the level of employment and unemployment, the level of the social economy, laws, regulations, and the social security system. Microcosmic factors include the factors of organization, the individual, and so on. Research on the influencing factors of turnover intention among health care staff has mostly focused on individual characteristics, attitudes and behaviors; examples include personality traits, life traits, job satisfaction, job performance, burnout, and work stress. Hayes, based on a review of a large number of studies, attributed the factors affecting nurses’ turnover intention to organizational factors, workload, stress, burnout, management, empowerment, and individual factors . During However, previous studies have ignored the uniqueness of the health care industry; compared with other fields, the health care sector is a social welfare industry that has the attributes of a public service. Perry and Wise developed the concept of public service motivation (PSM) to understand the behavior of public organizations and the management of staff, such as public hospitals . TherefoSome scholars believe that employees with high levels of PSM may seek specific professions, especially the medical profession, whose aim is to help others . For theWe therefore proposed the following:Hypothesis 1 (H1):PSM has a negative effect on turnover intention.Most studies define work stress as a psychosomatic emotional response that arises from a mismatch between job demands and one’s abilities, resources and needs , 31. HeaBased on the above discussion, we formulated the following hypotheses:Hypothesis 2 (H2):Work stress has a positive effect on turnover intention.Hypothesis 3 (H3):PSM has a negative effect on work stress.Hypothesis 3a (H3a):Work stress mediates the relationship between PSM and turnover intention.Many studies on motivation indicate that while some people are selfish and driven by material things, others are moved by the identities and experiences of others . Nurses,Hence, we made the following assumptions:Hypothesis 4 (H4):Task performance has a negative effect on turnover intention.Hypothesis 5 (H5):PSM has a positive effect on task performance.Hypothesis 5a (H5a):Task performance mediates the relationship between PSM and turnover intention.Hypothesis 6 (H6):Work stress has a negative effect on task performance.Hypothesis 6a (H6a):Task performance mediates the relationship between work stress and turnover intention.We collected the data using a questionnaire survey among medical staff in public hospitals in Jilin Province, China. First, we divided all public hospitals in Jilin Province into urban and county-level public hospitals. Because of the high clustering of urban public hospitals, we applied stratified random sampling to select the sample of urban public hospitals on a 1/4 scale stratified by region, type and level. At the county level, we chose the People’s Hospital and the Traditional Chinese Medicine Hospital as the sample of hospitals from each county. After this process, we finally included a total of 109 hospitals, including 80 county-level public hospitals and 29 urban public hospitals. Next, we sampled 30 medical personnel, including doctors and nurses, from each hospital using quota sampling. Finally, trained investigators conducted an on-site questionnaire survey of medical personnel and recovered the questionnaire on the spot. Our study was approved by the Medical Ethics Committee of the authors’ institute (No. 2019-12-03). In addition, during the survey, the investigator fully explained to the medical personnel the purpose of the survey and informed them that they could withdraw at any time.A total of 3260 questionnaires were distributed, and after removing the unqualified questionnaires, 3191 valid ones were retrieved, with an effective recovery rate of 97.88%.We adapted validated scales from previous studies to measure the variables.We obtained items for work stress from the Challenge-and Hindrance-Related Self-Reported Stress Measures (CHSS) developed by Cavanaugh et al . The CHSWe based items for task performance (TP) on the performance measurement scale . TP contWe obtained the measurement items from the PSM scale developed by Perry, which is widely used by scholars , 47. In I had the idea to leave the hospital,” “Within a year, I will look for a new job,” and “If I had the chance, I would definitely accept a better new job.” We measured each item using a 5-point Likert scale, and the higher the score, the stronger the turnover intention of the medical personnel. In this study, the coefficient was 0.849.Most of the turnover intention measurements refer to the scale in Mobley’s employee withdrawal behavior model . Based oIn addition, for the questionnaire we collected the demographic characteristics of the respondents, including gender, age, marital status, education level, professional title, department, and working years.We combined descriptive statistics, t-tests, one-way analysis of variance (ANOVA), structural equation modeling (SEM), and mediating effects in the analytical strategy. We used descriptive statistics to analyze the demographic characteristics of the medical personnel, including gender, age, marital status, education level, professional title, department, and working years. We applied t-tests and ANOVA to describe the differences in the turnover intention of respondents with different characteristics. We used Pearson’s correlation analysis to test the correlations among the variables. We employed Amos 24.0 to construct a model to test the hypotheses. We used the maximum likelihood method in the parameter estimation. We adopted the following criteria to evaluate the model’s fitness , 52:(1) the chi-square test;(2) root mean square error of approximation (RMSEA ≤ 0.08);(3) the comparative fit index (CFI ≥ 0.90);(4) the Tucker Lewis Index (TLI ≥ 0.90);(5) the incremental fit index (IFI ≥ 0.90);(6) the normed fit index (NFI ≥ 0.90); and(7) the standardized root mean-square residual (SRMR ≤ 0.05).Finally, we analyzed the mediating effects of work stress and task performance using the bootstrapping technique. All statistical tests were two-sided, with the level of significance set at 0.05.Of the 3191 participants, 69.73% (n = 2225) were women, and the mean age was 37.54 years (SD = 8.86). The majority were married . Approximately 69.10% (n = 2205) had a college degree or higher. Most participants had junior or middle professional titles , and only 4.51% (n = 144) had senior professional titles. These medical personnel were mainly from the departments of internal medicine or surgery, and had worked in hospitals for an average of 12.5 years (SD = 9.48). The demographic characteristics of the participants are shown in Table The turnover intention of the participants was 2.02 ± 1.13. The difference in turnover intention between males and females was statistically significant, and the turnover intention of males (2.24 ± 1.20) was higher than that of females (1.93 ± 1.09). The turnover intention of medical staff in different departments was also significantly different, and medical personnel from pediatric (2.14 ± 1.18) and surgical (2.13 ± 1.18) departments had higher turnover intentions than those from other departments. However, there were no significant differences in turnover intention for age, marital status, education level, professional title, or years of work experience. The turnover intention scores of medical personnel with different characteristics are shown in Table r = −0.362, p < 0.01) and turnover intention  and positively correlated with task performance . Work stress was positively correlated with turnover intention  and negatively correlated with task performance . Task performance was negatively correlated with turnover intention .The scores of work stress, task performance, and PSM are shown in Table t and p values for each path. Table We established a structural equation model to verify the theoretical hypothesis and to explore the interrelationships among the variables. We applied maximum likelihood to fit the data, and we calculated the β = −0.294, P < 0.001), which meant that the higher the PSM, the lower the turnover intention of medical personnel. However, work stress directly and positively affected turnover intention , indicating that the greater the work stress, the higher the turnover intention. Therefore, both H1 and H2 were supported. Although the P of the path coefficient for task performance and turnover intention was less than 0.05, whereas it was close to 0.05, and the 95% CI of Bias-Corrected method and Percentile method showed that their confidence intervals contained 0. Therefore, we thought that task performance did not have a significant influence on turnover intention, so H4 was not supported. In addition, the higher the PSM, the lower the work stress, and the better the task performance , so H3 and H5 were supported. The higher the work stress, the worse the task performance , so H6 was also supported. As shown in Fig. PSM directly and negatively affected turnover intention , whereas the mediating effect of task performance was not significant . In addition, the effect of work stress on turnover intention through task performance was not statistically significant. Therefore, H3a was supported, whereas H5a and H6a were not. Further, the total effects of PSM and work stress on turnover intention were −0.470 and 0.473, respectively. Table With regard to the mediating effects, we adopted the bootstrapping technique in AMOS to explore the mediating role of work stress and task performance, and we obtained the 95% confidence intervals of the indirect effects with 5000 bootstrap resamples. PSM significantly affected the turnover intention of medical personnel through work stress  is basically consistent with the results of other studies focusing on the turnover intention of health care workers in China. For example, Ting Cao et al. (2020) reported a turnover intention score of 2.21 (SD = 0.75) in a study of 569 registered nurses in Beijing, China .First, work stress positively affected the turnover intention of health care workers. Higher work stress leads to organizational inefficiency, absenteeism due to illness, increased health care costs, and a high turnover of staff . This arSecond, the turnover intention of health care workers is influenced by PSM, and the turnover intention of health care workers with higher PSM is lower . SimilarHowever, the task performance of medical staff has no effect on turnover intention, which may be related to the characteristics of medical work. Medical work has unified rules and regulations, operating norms and clinical practice guidelines, and medical personnel are strictly required to follow the correct steps and norms for practical work. Andersen’s (2009) research supports this inference, and the performance and behavior of health care workers managed by professional norms are similar, even when they have different incentives .We propose exploring the essential meaning of medical services, improving medical education, inspiring a sense of morality and compassion among medical staff, and enhancing their level of PSM to reduce the turnover intention. There are few studies on the turnover intention of medical staff from the perspective of public management, so this paper can fill the gap to some extent. In addition, the shortage of, and increasing pressure on, medical staff are the two most serious problems against the background of coronavirus epidemic prevention and control. Our results are more widely applicable than those of other studies that focus on a single population, can provide a reference for hospital human resource management from a comprehensive standpoint, and have strong application significance. However, we have to admit the limitations of this paper. First, this study was limited to public hospitals in Jilin Province, so the results may not be generalizable. Second, the study was a cross-sectional one and was unable to reflect the causal relationship between variables.Given the severe shortage of health care workers worldwide, we explored the relationship between turnover intention and PSM, task performance, and work stress in Chinese public hospitals. We found that the greater the PSM, the lower the turnover intention, whereas the higher the work stress, the higher the turnover intention. In addition, work stress and task performance play intermediary roles between PSM and turnover intention. This paper provides a useful reference for creating measures to reduce the turnover intention of medical staff.


Olfactory-induced emotion plays an important role in communication, decision-making, multimedia, and disorder treatment. Using electroencephalogram (EEG) technology, this paper focuses on (1) exploring the possibility of recognizing pleasantness induced by different concentrations of odors, (2) finding the EEG rhythm wave that is most suitable for the recognition of different odor concentrations, (3) analyzing recognition accuracies with concentration changes, and (4) selecting a suitable classifier for this classification task. To explore these issues, first, emotions induced by five different concentrations of rose or rotten odors are divided into five kinds of pleasantness by averaging subjective evaluation scores. Then, the power spectral density features of EEG signals and support vector machine (SVM) are used for classification tasks. Classification results on the EEG signals collected from 13 participants show that for pleasantness recognition induced by pleasant or disgusting odor concentrations, considerable average classification accuracies of 93.5% or 92.2% are obtained, respectively. The results indicate that (1) using EEG technology, pleasantness recognition induced by different odor concentrations is possible; (2) gamma frequency band outperformed other EEG rhythm-based frequency bands in terms of classification accuracy, and as the maximum frequency of the EEG spectrum increases, the pleasantness classification accuracy gradually increases; (3) for both rose and rotten odors, the highest concentration obtains the best classification accuracy, followed by the lowest concentration. Emotion is an ever-changing psycho-physiological phenomenon that represents the body’s adaptation to environments . As a hiAt present, for the induction of emotions, researchers generally use visual, auditory, or a combination of visual and auditory stimuli ,7,8. Howk-nearest neighbor (k-NN) [Many techniques can capture the emotion state under olfactory stimulation ,17,18,19r (k-NN)  methods As shown above, the research on olfactory EEG recognition has made significant progress. However, all the presented studies use only different odor types to stimulate the participants. To the best of our knowledge, the research on emotion recognition caused by different concentrations of odors is currently scarce. For stimulus studies of odor concentration, Tateyama et al.  exploredThis article explores pleasantness recognition induced by different odor concentrations for the first time. The main purpose of this study is (1) to explore the classification possibility of odor concentration-induced emotions using EEG signals, (2) to find the best EEG rhythm wave for this recognition task, (3) to analyze the relationship between recognition accuracy and odor concentrations, and (4) to select the most suitable one among five commonly used classifiers.The structure of this paper is as follows. In rose odor, and the other is a foul, unpleasant odor, termed rotten odor, and each includes five different concentrations. The five concentrations of each odor type from low to high are described as A10−5.0, A10−4.5, A10−4.0, A10−3.5, and A10−3.0 and C10−6.0, C10−5.5, C10−5.0, C10−4.5, and C10−4.0, respectively, where the values 10−3.0, 10−3.5, …, 10−6.0 indicate the dilution coefficients of the concentration. For different odor types, the central concentration, e.g., A10−4.0 or C10−5.0, which corresponds to the detection threshold of normal olfactory sensation, may be different. If someone can smell the central concentration, it means that their sense of smell is normal; otherwise, it is abnormal. Because of the different central concentrations of different odor types, the magnitudes of the exponents  of rotten and rose odors are different.Two kinds of odors in the T&T olfactometer  were used; one smells like roses, termed During the experiment, the smells were randomly selected and placed 1–2 cm below the participant’s nose through test papers. Subsequently, a Cerebus system  categories and 350 (10 × 35) EEG samples. In total, 4550 (350 × 13) EEG samples were collected.The high-frequency noise of EEG signals was removed using a 0.5–70 Hz bandpass filter, and then the power spectral density (PSD) features were calculated in each of the five typical EEG bands, namely, delta (δ): 0.5–3 Hz, theta (θ): 4–7 Hz, alpha (α): 8–13 Hz, beta (β): 14–30 Hz, and gamma (γ): 31–70 Hz.k-NN algorithm [In the classification stage, we compared five classical and commonly used classifiers, such as SVM , naive Blgorithm , extremelgorithm , and baclgorithm , in whicIn the experiment, each participant performed a subjective evaluation of the pleasure level induced by each odor concentration. For each concentration of each odor, the subjective score ranged from −10 to 10. Positive scores represent pleasant emotions, where greater values represent more pleasant odors; the negative scores represent disgust emotions, where greater absolute values indicated more disgusting odors. The subjective evaluation scores are shown in The results show that different concentrations of odors can induce different degrees of pleasure or disgust. To obtain emotional categories, the average subjective evaluation scores of 13 participants were calculated. As a result, five different levels of pleasant emotions can be induced by the five different concentrations of rose odor, while five different levels of disgust emotions can be induced by the five different concentrations of rotten odor.p-values) for the rose and rotten odors are 4.7 × 10−3 and 6.2 × 10−8, respectively. Both of these are less than the significance level of 0.05, proving that for the subjective evaluation scores of 13 participants used in this study, the differences between the five concentrations of each odor type can reflect the overall differences between the odor concentrations. In other words, the significance test results prove that the levels of emotion vary with concentrations.To validate the claim of varying levels of emotions with concentrations, statistical tests, specifically, the Friedman significance test, for the subjective evaluation scores of five concentrations of each odor type have been performed, and the significance test results  EEG usually has limitations and it is better suited to MEG studies. However, for olfactory EEG, from the perspective of signal recognition, we found that the features of gamma band are better than those of other EEG rhythms, which has also been proved by other scholars . MoreoveFor the recognition methods, the current research only adopted and verified the feasibility of several classical methods. However, this study does not focus on the method innovations, but the innovation of research content, that is, recognizing emotions induced by different odor concentration through EEG analysis. As far as we know, there are few reports about inducing people’s emotions through different odor concentrations and then conducting such emotional recognition through EEG analysis.Although the experiments in the literature e.g., ,24,28) a,28 a24,2Considering that the focus of this study is to test the feasibility of recognizing emotions induced by different odor concentrations through EEG, at this stage, we have not compared the adopted methods with those in other papers. In subsequent research, we will design novel recognition algorithms with better performance for this specific recognition tasks, and compare it with other methods in the literature.Moreover, by considering and comparing our results with previously reported studies in the literature, the novelty of our study is expressed as follows: The detailed contribution points and the main purpose of this study include: (1) to explore the classification possibility of odor concentration-induced emotions using EEG signals, (2) to find the best EEG rhythm wave for this recognition task, (3) to analyze the relationship between recognition accuracy and odor concentrations, and (4) to select the most suitable one among five commonly used classifiers.Taking the gamma frequency band as an example, to explore the maximum of the PSD amplitudes among all electrodes, which is helpful for the determination of the brain regions related to the sense of smell, we tested each odor concentration and obtained the average PSD amplitudes of 35 EEG samples corresponding to each concentration for each participant. The study found that for different concentrations of the same odor type, the electrode corresponding to the maximum of the PSD amplitudes was the same. However, there are differences in the electrodes corresponding to the maximum amplitude of the PSD of the two odor types. Size of participants: Compared to datasets with a small number of participants, large datasets are more powerful to support the research. The sample size (13 participants) used in our work is small, but we think that the EEG signals collected from 13 participants can also be used to carry out exploratory research of pleasantness recognition induced by different concentrations of odors. Therefore, in this study, the dataset involving 13 participants was presented, which has been openly available on the website . To furtGender non-homogeneity: For the gender ratio of participants, this study involved 13 participants, including 12 men and 1 woman, which is gender non-homogeneity. For this issue, we will increase the number of female participants in the following research, and maintain gender balance while expanding the scale of participants.The training process in our study contained five steps. (1) Randomly select training samples; (2) Extract features from the selected samples; (3) Feed features into the classifier to determine classifier parameters; (4) Use the determined classifier to predict the categories of new EEG samples; (5) Repeat the presented steps 10 times to obtain the average prediction accuracy of 10 times. The results in −4.0; −5.5, thus proving that the concentrations/emotions can be effectively predicted.When given the EEG signals, the concentrations/emotions can be effectively predicted. The accuracies shown in At present, this study is only a preliminary exploration of emotion recognition induced by different odor concentrations. At this stage, we have tried to analyze the emotions induced by only pleasant and disgusting odors. Through the preliminary exploration, the feasibility of recognizing olfactory-induced emotions through EEG analysis has been verified.For recognition of emotions induced by different odor concentrations using EEG signals, the T&T olfactometer which contains five odor types, each with different concentrations, provides a very suitable odor material. After the psychological evaluation, we eliminated the intolerable sweat odor and feces odor, and finally screened out two odor types, one being the pleasant rose odor, the other the disgusting rotten odor, each odor with five different concentrations. The next research will add the analysis of neutral odor-induced emotions according to your valuable comments.k-nearest neighbor, extreme learning machine, and backpropagation neural networks, the support vector machine demonstrates the feasibility for recognizing the odor-induced pleasantness.This study uses EEG technology to recognize pleasantness induced by different concentrations of odors. Using the support vector machine with power spectral density features of the EEG gamma frequency band, the average classification accuracies of 93.55% and 92.24% were achieved for the pleasantness recognition induced by different concentrations of pleasant rose and disgusting rotten odors, respectively. When classifying the pleasant and disgusting emotions, a considerably high accuracy of 99.9% was obtained. The experimental results reveal that (1) it is possible to classify pleasantness emotions induced by odor concentrations; (2) in the EEG rhythm, the gamma band is most related to odor concentration stimuli; (3) compared to the naive Bayesian,


Bifidobacterium breve HA-129, Lacticaseibacillus rhamnosus HA-111, Bifidobacterium bifidum HA-132, Bifidobacterium longum subsp. infantis HA-116, and Bifidobacterium longum subsp. longum HA-135 (FloraBABYⓇ) daily until discharge or transfer from hospital. The incidence of NEC was compared among infants for 2 years pre- and post implementation of the protocol then 6-years following continuous implementation of the probiotic use. In total, 370 infants not treated with probiotics between 2012 and 2014 were included with an incidence of NEC at 4.9%. In comparison, the 367 infants who received had a 67% reduction  in our Neonatal Intensive Care Unit (NICU). The results remained significant  after adjusting for GA, small for gestational age, and antenatal corticosteroid use. Data from the Canadian Neonatal Network not only showed a consistently high rate of NEC in October 2014, but also identified exceedingly high rates (8.7–15.6%) in some hospitals up to 2021, while our rates have been consistently low with using the probiotic as standard therapy for low BW premature babies, with no serious side effects reported. In conclusion, the introduction of a five-strain probiotic natural health product has coincided with a reduced incidence and complications of NEC in our NICU setting.The objective of this retrospective cohort study was to assess the impact of an enteral probiotics supplementation protocol on the incidence of necrotizing enterocolitis (NEC) in infants born <33 weeks gestational age (GA) or birth weight (BW) <1,500 g. In addition, a 6-year follow-up is presented after instigation of probiotic use. In October 2014, our NICU introduced an enteral probiotics supplementation protocol for infants born <33 weeks GA or BW <1,500 g. Infants received 0.5 g of  Necrotizing enterocolitis (NEC) is one of the most devastating and unpredictable gastrointestinal emergencies in very low birth weight (VLBW) infants , 2. The The precise etiology of NEC remains unclear but appears to be multifactorial including: 1) immaturity of host defenses, (2) prematurity, (3) intestinal ischemia, (4) imbalances of inflammatory responses, and (5) abnormal bacterial colonization and pathogen overgrowth  immaturi, 9. The In term babies, lactobacilli are present followed by bifidobacteria that utilize human milk oligosaccharides to propagate . In ordeLimosilactobacillus reuteri in extremely low birth weight infants  reduced the incidence from [35/232 (15.1%) to 2/79 (2.5%)] using a historical cohort  demonstrated a significant reduction in the incidence of NEC despite different probiotic strains being used . The usel cohort .Ⓡ, a multi-strain probiotic product containing 0.5 g of Bifidobacterium breve HA-129 [600 million colony forming units (CFU)], Lacticaseibacillus rhamnosus HA-111 (500 M), Bifidobacterium bifidum HA-132 (400 M), Bifidobacterium longum subsp. infantis HA-116 (300 M), and Bifidobacterium longum subsp. longum HA-135 (200 M) was 3-fold: (i) They are manufactured in Canada and approved as a Natural Health Product, abiding by Good Manufacturing Practice (GMP); (ii) They have been shown to be safe and effective for eligible infants at <32 weeks gestation, lowering the incidence of NEC at Sainte Justine University Health Centre, in Montreal, Canada  T, Canada .Ⓡ , to infants admitted after implementation , and found a significant decrease in NEC from 9.8 to 5.4% with no reports of adverse effects .Patients admitted to the NICU at the Children's Hospital at London Health Sciences Centre between September 1, 2012 and July 31, 2014 were included in the pre-probiotics cohort, and patients admitted between October 1, 2014 and December 31, 2016 were included in the post-probiotics cohort. All infants admitted in August and September of 2014 were excluded to account for a washout period when probiotics use was permitted with parental consent but was not yet a routine practice for all eligible infants. All patient medical information was extracted from our local neonatal-perinatal database. This study was approved by the Western University Ethics Review Board.Ⓡ  probiotics mixed with 1–2 mL of sterile water immediately prior to commencing a feed and administered via NG/OG or PO once daily. Probiotics were started at the time of the first feed and continued until the infant was discharged from the NICU or transferred to another institution. The probiotic was continued throughout episodes of acute illness unless the infant was placed nil per os (NPO); in such case, probiotics were withheld, and reintroduced following the period of NPO at the discretion of the attending neonatologist.Infants <33 wks GA, or BW <1,500 g, received 0.5 g FloraBABYThroughout the study period, infants' nutritional needs were managed with a specific institutionalized feeding protocol, which preferentially favored breastmilk and early enteral feeding in preterm infants. The use of donor human milk selectively commenced in November 2012. Over the subsequent years, the donor human milk program underwent expansion of eligibility criteria until finally, in February 2015, the program expanded to include all infants <34 weeks GA. Other than this expansion of the eligibility criteria, the feeding protocol remained unchanged and no other practice changes occurred.The primary outcome of this investigation was the development of definite NEC, diagnosed based on combined clinical and radiological evidence, and classified according to the Bell classification system . For theSecondary outcomes included: (1) mortality , (2) sepsis , (3) number of days to full enteral feeds, (4) retinopathy of prematurity according to the international classification , (5) intt-tests and chi-square tests for continuous and categorical outcomes, respectively. Logistic regression models were used to examine differences between pre- and post-probiotic cohorts while controlling for GA, small for gestational age (SGA), and the use of maternal antenatal corticosteroids (ACS). SPSS v.25  was used for all analyses, and p-values < 0.05 were considered statistically significant.Population demographics were analyzed using frequencies and descriptive statistics. Pre- and post-probiotic cohorts were compared with The Canadian Neonatal Network™ collects data from 33 Health Care Organizations, publishing an annual report that includes the incidence of NEC and related mortality. We acquired data that is based on calendar year, and thus the incidence of NEC is marginally higher for 2012 to 2014 (5.1% compared to 4.9% which we report herein) as we covered September 2012 to October 2014 for our rate.A total of 845 infants <33 weeks GA or BW <1,500 g were admitted to the NICU between September 1, 2012, and December 31, 2016, and assessed for eligibility; 108 were excluded for various clinical complications or parental input, and 737 infants were included in the final analyses: 370 in the cohort assessed without probiotic use, and 367 who received the probiotic . Baselinp = 0.009) after controlling for GA, SGA, and ACS.A significant decrease in the incidence of definite NEC was observed in the cohort receiving probiotics compared to the babies who had not received the supplement, from 4.9 to 1.6%  after controlling for GA, SGA, and ACS , intraventricular hemorrhage (IVH), patent ductus arteriosus (PDA), bronchopulmonary dysplasia (BPD), or the number of days to achieve full enteral feeding .The data from CNN was informative in terms of showing that the incidence of NEC was on average lower than the other hospitals post-probiotic introduction in London (2.01 v 3.67) . HoweverGiven the substantial mortality and long-term morbidity associated with NEC, prevention of this disease has become a priority in the management of preterm, VLBW neonates. Although the exact etiology of NEC remains unclear, pathogenic bacterial colonization is thought to be a critical factor in the disease development. As such, the application of probiotic bacteria to the gut has been shown to be feasible and efficacious as a prevention strategy , 28. TheBased on the present results, the practice change we underwent in 2014 has been justified. The results were consistent with the existing body of literature, as we saw a 74% reduction in the odds of developing NEC, and a trend toward reduction in mortality after controlling for GA, SGA, and use of ACS in infants born <33 weeks GA or with BW <1,500 g. A study using the same probiotic formulation showed NEC ≥ Bell Stage 2 reduced from 0.14 to 0.04 per 100 patient days, as illustrated in a U chart .The mechanisms whereby these probiotic strains confer the benefits is still unclear but may be through enhancing a protective effect of mucosal barrier integrity, competitive exclusion of pathogens, modification of host response to microbial products, augmentation of IgA mucosal responses, enhancement of enteral nutrition that inhibit the growth of pathogens, and up-regulation of immune responses . The impDespite the strong evidence favoring the use of probiotics in NEC prevention, there remains to be widespread uptake and use amongst neonatal care providers. Providing live microorganisms to an immunocompromised host creates hesitation and raises concern regarding the risk-benefit profile. This risk is of particular concern in preterm infants given the immaturity of their intestinal barrier and increased risk of translocation of intestinal microbes into the systemic circulation . AlthougN = 7,345 infants) showing multi-strain probiotics reduced the odds of NEC by 64%  and mortality by 42% , whereas single strains had a borderline effect in reducing the odds of NEC  but not mortality. The hypothesis is that multiple strains provide increased diversity of the gut microbiota and reduce colonization by pathogenic organisms. However, the evidence for this is limited and large comparative trials would be required to confirm it.The present study used a multi-strain product, but we cannot conclude that it would be better than a single strain as Chang et al. have suggested  based upThe development of the premature gut microbiota is known to be influenced by multiple factors including GA, use of ACS, mode of delivery, antibiotic exposure, and diet. We were unable to assess the diet of enrolled infants (proportion human milk vs. formula) in the two cohorts; however, our standard feeding protocol, the rate of breastfeeding initiation (>90%), and percentage of mother's own milk intake (60–70%) remained unchanged during the study period. The criteria for donor human milk were expanded throughout the study period to be more inclusive, and it is likely that the post-probiotic cohort received more donor milk  than the pre-probiotic cohort. In fact, the concern has been raised that increased use of donor human milk may confound the results of probiotics studies. However, the ProPrems Trial , in whicThere are limitations to our study. Misclassification bias is inherent in retrospective studies, and the expanded use of donor milk to higher GAs could potentially have led to an overestimation of our calculated effect size. Nevertheless, as shown from long term follow-up, the standard administration of a five-strain probiotic has allowed retention of a low rate of NEC amongst the ever-increasing VLBW infants admitted to our unit. Given the high rates that still occur in hospitals, and the long-term ailments that occur , NICUs sThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Western University Ethics Review Board. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.GR and OS initiated the study. BB, EH, CU, and OS recruited the patients and acquired the data. MM performed the statistical analysis. BB, GR, and OS wrote the manuscript. All authors contributed to the article and approved the submitted version.


Although pediatric urolithiasis is an established entity, its antenatal diagnosis is rare. We hereby report a case detected at 20 weeks gestation and discuss the etiopathogenesis, predisposition, and surveillance following intervention.A 2-year-old girl with left renal pelvic calculus detected antenatally at 20 weeks was evaluated. Left hydronephrosis, obstructive pelvic calculus with a decrease in differential renal function on ethylene dicysteine (EC) renogram was confirmed. The metabolic workup was normal. Following stone extraction by left pyelolithotomy, a left ureteropelvic junction obstruction secondary to a mucosal valve was apparent which was excised and left pyeloplasty was done. Stone analysis revealed 100% cystine. Differential renal function and drainage improved post-surgery. The child, however, did not have a follow-up in the interim and presented with a recurrent stone one and a half years later.Knowledge of antenatal urolithiasis ensures continued follow-up, evaluation for metabolic disorders, and associated structural defects, especially with increasing stone size and increasing hydronephrosis. This helps in timely intervention and continued surveillance. Although 20–30% of all the prenatally identified anomalies relate to the urinary tract , antenatA 2-year-old asymptomatic girl, was referred for the management of left renal calculus. Antenatal ultrasound at 20 weeks gestation had multiple echogenic foci in the left lower calyx which persisted at 28 weeks with normal amniotic fluid volume. She was born full-term, weighed 2.8 kg, and had an uneventful perinatal period. Renal ultrasound at 6 months showed 2–3 hyperechoic foci in the lower pole of the left kidney, with the largest being 6.2 mm with a pelvic diameter of 9 mm, spot urine calcium (8.1 mg/dl), urine creatinine (17.59 mg/dl), and calcium/creatinine ratio of 0.46. At 1 year of age, serum creatinine was 0.38 mg/dl (0.6–0.9 mg/dl). Serum calcium (10.8 mg/dl) , phosphorous (6.6 mg/dl) , magnesium (2.5 mg/dl) , alkaline phosphatase (222 U/L) (81–350 U/L), and uric acid (3.5 mg/dl) (2.5–5.5 mg/dl) were normal. She was referred to us for further management due to a progressive increase in the size of the calcific foci with increasing hydronephrosis. At 2 years, she weighed 9.3 kg and 81 cm in height, with a blood pressure of 100/70 mmHg (99 percentile). Her hemoglobin, electrolytes, serum bicarbonate, and renal functions were normal but eGFR was 73 ml/min- . Ultrasound showed a left renal pelvis diameter of 13 mm with a 2 cm stone in the pelvis . The rigIn view of the large size stone, endoscopic management was deferred. A 3 cm × 3 cm calculus, partially intra- renal extending into the lower calyx was retrieved by a left pyelolithotomy. On probing the ureter, a mucosal valve causing narrowing at the ureteropelvic junction was noted . This paIncidence of urolithiasis is between 1 in 3,000 and 1 in 8,000 of all pediatric admissions and is 10% of that in adults . PediatrThe first case was a 28-week monochorionic, diamniotic twin gestation, and mild polyhydramnios with one of the twins having a 3-mm echogenic focus in the upper part of the right kidney. At 3 month postnatal ultrasound, it was revealed that there were multiple right upper pole stones with the largest measuring 5 mm. The baby was asymptomatic with normal metabolic workup and VCUG and was well up to 6 months follow-up .Rhodes et al. reported antenatal urolithiasis detected at 34 weeks gestation as an echogenic focus in the upper pole of the right kidney which persisted up to 4 months. No metabolic or structural abnormalities were noted in the child during follow-up . DetectiUrinary stones are composed of lithogenic crystal agglomerations and are formed on the renal papillae by adherence to the damaged renal epithelium. The imbalance of the activating factors such as high calcium/oxalate and the inhibitory factors such as low citrate excretion dictates stone formation. Almost 50% of pediatric urolithiasis cases are idiopathic, and 30 and 20% are due to hypercalciuria and hyperoxaluria, respectively. Rarely hyperuricosuria, xanthinuria, and hypocitruria are the cause . Almost A 2-day-old male neonate with gross hematuria and a 3–4 mm calculus in the upper pole of the right kidney with no metabolic or structural abnormality was managed conservatively , whereasAs in our child, who was asymptomatic but had persistence of stone with a gradual increase in size and normal metabolic parameters, a thorough evaluation for a structural abnormality is stressed. Following stone retrieval, probing of the ureter for patency is also emphasized so that an occult obstructive pathology is not missed. In the present case, the valve seems to be a result of mucosal inflammation caused by the stone present since the prenatal period. A 100% cystine on stone analysis confirms Cystinuria.Cystinuria is an autosomal recessive disorder, characterized by failure of reabsorption of cystine and other dibasic amino acids in the renal tubules . CystinuAmat S. et al. concluded that the presence of a hyperechoic colon at a routine ultrasound scan before 36 weeks gestation should prompt screening for cystinuria at birth, while later observation (> 36 weeks) does not relate to any disease . In our The current case is one of the rare occurrences of prenatal urolithiasis thus far reported in the literature. Knowledge of antenatal urolithiasis helps in planning the most appropriate treatment. Emphasis is on prolonged follow-up and evaluation for associated structural defects and metabolic disorders, especially in cases with increasing stone size and increasing hydronephrosis. This will prompt a timely surgical intervention, initiation of appropriate medical treatment, and regular follow-up, all aiming to preserve renal function.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Institutional Ethics Committee, St. Johns Medical College and Hospital, Bangalore. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.SB contributed towards conception and design, data collation, analysis and interpretation of data, drafting of the manuscript, and involved in obtaining ethical approval. AS and AI contributed toward study design, patient management, data collation, drafting, critical revision and editing of the manuscript, and supervision at all levels. All authors have reviewed and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Glomerulopathy with fibronectin deposits (GFND) is a rare autosomal dominant genetic disorder, and proteinuria and hematuria are the most common clinical manifestations. The pathogenesis of this disease is primarily related to mutation of the fibronectin 1 gene. Unfortunately, without specific treatment, the prognosis remains poor. Here we present a case report that investigates the clinical characteristics, renal pathology, and gene testing of childhood GFND.A two-year-old child was brought to our hospital for “persistent hematuria for 1 year and 10 months.” The disease onset was at the age of 4 months, with persistent microscopic hematuria accompanied by intermittent gross hematuria, occasionally with proteinuria, and without hypertension or renal failure. The chief complaint was intermittent gross hematuria, without massive proteinuria, hypertension, or renal failure. Family history: The child’s mother had microscopic hematuria, his maternal aunt had nephrotic syndrome due to focal segmental glomerulosclerosis, and his maternal grandmother had end-stage renal disease. No significant pathological changes were found in the renal pathological biopsy of the child under a light microscope. Under the electron microscope, the basement membrane was found to be of uneven thickness, ranging from 150 to 400 nm. The stratum compactum of the basement membrane was thickened, with a small part showing tear-like and cobweb-like morphology. No electron-dense deposits were found. The renal tubular epithelial cells were vacuolated, and there were no unique pathological changes in the renal interstitium. Immunofluorescence showed that IgG, IgM, IgA, C3, and C1q were all negative. Alport syndrome was preliminarily considered. However, exome sequencing revealed a mutated site in the fibronectin 1 gene. The child’s mother was the carrier of the pathogenic gene and the final diagnosis was GFND.Fibronectin deposition is a typical pathological change in GFND, and the disease progresses slowly to end-stage renal disease. There is no specific treatment so far, and the prognosis is poor. The early onset of childhood patients may not show typical renal pathological changes, but only changes in the thickness of basement membrane, etc. Genome sequencing technology may helpful for the early diagnosis of GFND. Glomerulopathy with fibronectin deposits (GFND) is an autosomal dominant glomerulopathy with age-related penetrance, characterised by fibronectin (FN) deposits within the glomeruli, the clinical manifestations of which are mainly proteinuria, microscopic hematuria, and hypertension , 2. The A boy, aged 2 years and 2 months, was admitted to our hospital on August 31, 2016, due to “microscopic hematuria found 1 year and 10 months ago, and intermittent gross hematuria for 2 months.” Microscopic hematuria was found during a health examination at the age of 4 months. Examinations showed: Urine protein 2+, occult blood 2+, urine microalbumin ≥ 100 mg/L. Urinary infection was initially diagnosed and Mezlocillin and sulbactam were administered for 5 days as an anti-infective therapy, and the hematuria improved upon reexamination. At the age of 6 months, the child underwent examinations, in which urine routine test showed 12–14 red blood cells/HP. Two months ago, the child intermittently discharged dark tea-colored urine. Urine routine test showed occult blood 3+. The child was referred to our hospital for further diagnosis and treatment. Physical examination showed no abnormal changes in the vision and hearing systems were observed in this child.Family history was inquired upon admission: The child’s maternal grandmother had kidney failure by the age of 50 years, his maternal aunt had nephrotic syndrome with focal segmental glomerulosclerosis revealed by renal pathological biopsy at the age of 20, and his mother had a history of hematuria and nephrolithiasis for 5 years.Laboratory examinations upon readmission included: Urine routine test: Urine occult blood 3+, red blood cells 1428.5/ul, protein (-); Urine red blood cell phase: Red blood cells 1,400,000/mL, percentage of malformed red blood cells 90.0–95%; blood albumin 36.7 g/L, total cholesterol 5.47 mmol/L, triglyceride 0.69 µmol/L, urea nitrogen 4.3 mmol/L, creatinine 27.6 µmol/L; 24-hour proteinuria 0.047 g/24 h, urine microalbumin 1.78 mg/dl. Coagulation function,immunoglobulin, complement, erythrocyte sedimentation rate and stool routine were normal; the 5 markers for hepatitis B, mycoplasma pneumoniae, EBV, and tuberculosis antibody were all negative. Color Doppler ultrasonography showed that the size of both kidneys was normal.On the 6th day after admission, renal puncture biopsy was performed under the guidance of B-mode ultrasonography. Pathological results of renal biopsy included light microscope findings: Routine HE, PAS, PASM, and Masson staining was performed on the renal puncture biopsy tissue. A total of 18 glomeruli were observed. The mesangial cells and matrix did not proliferate significantly. There was no significant deposition of fuchsinophilic protein in the mesangial, subepithelial, or subendothelial region. The capillary loops were open, the basement membrane was not significantly thickened. Granular degeneration of renal tubular epithelial cells was found without significant atrophy. No significant inflammatory cell infiltration or fibrosis was found in the renal interstitium. No significant pathological changes of the arteriole walls were found. Electron microscope findings: The capillary endothelial cells were significantly vacuolated. Red blood cells could be seen in individual lumens. No significant endothelial cell proliferation was found. The capillary loops were open. The parietal layer of Bowman’s capsule was not significantly thickened or delaminated. The parietal cells were vacuolated. No significant hyperplasia was found; Basement membrane: The basement membrane was of uneven thickness, with a range of about 150–400 nm. The stratum compactum of the basement membrane was thickened, with a small part showing tear-like and cobweb-like morphology. Visceral epithelial cells: The epithelial cells were swollen and vacuolated, and phased fusion of foot processes was observed; Mesangial region: The mesangial cells and matrix had proliferated without electron-dense deposits. Renal tubules and interstitium: The tubular epithelial cells were vacuolated, while the renal interstitium was unremarkable; Renal interstitium blood vessels: Erythrocyte aggregation was seen in the lumens of individual capillaries, while no pathological changes were found in the arterioles. Immunofluorescence: IgG, IgM, IgA, C3, and C1q were all negative.Taking into consideration these findings, renal injury from Alport syndrome was considered. The pathology slides are shown in Fig. Alport syndrome, also known as hereditary nephritis, is characterized by hematuria, proteinuria, and progressive renal failure. Some patients may suffer from extrarenal symptoms such as sensorineural hearing loss and vision loss . The COLFirst reported by Tuttle et al. in 1987, GFND is a rare hereditary glomerulopathy newly recognized in recent years –8. As a The typical histopathological changes of GFND are glomerular lobular pattern, mesangial and GBM expansion, double contour and strongly positive fibronectin, and deposition of a large amount of fine fibrous materials can be observed under electron microscope. The child in this case had not shown the above-mentioned typical pathological changes yet. Considering that the child mainly manifested and proteinuria only occurred intermittently, the onset age of the child was young, the duration of the medical history was short, and typical pathological changes might not have occurred, it was suggested that long-term follow-up should be conducted for the child, and renal biopsy should be performed again as necessary to confirm the pathological diagnosis.The key pathogenesis of GFND is the deposition of fibronectin (FN) in glomeruli. At present, it is believed that the impaired ability of glomeruli to process deposits due to the FN1 gene defect is an important cause of GFND. The FN1 gene is located on chromosome 2 and the main mutation point. The abnormality of FN1 gene in this child was manifested by the substitution of cytosine by thymine in the base pair at position 2,984 of exon 19 [c.2984 C > T(exon19)] in the gene coding sequence of chromosome 2 (position: chr2:216270963); this caused the missense mutation of the amino acid at position 995 of the transcribed mRNA from threonine to isoleucine (p.T995I). This mutation simultaneously affects the interaction of the heparin-binding domain among fibronectin cells on the one hand and while promoting the formation of fibronectin on the other hand. In 2016, Ohstubo et al. [Currently, there is no effective or specific treatment for GFND, and only symptomatic treatment is available, in which hormonal and immunosuppressive therapies have no significant efficacy, with poor prognosis. Literature shows that even after kidney transplantation, the disease can still relapse . The chiThe limitation of this study is that, on the one hand, fibronectin and collagen were not stained in the biopsies of the children and their aunt, which affects the diagnosis of this disease to a certain extent. On the other hand, the impact of the disease on the children cannot be assessed without functional analysis of the detected mutations.In summary, GFND is an autosomal dominant genetic disease with familial inheritance. This disease should be considered when patients present with clinical manifestations of proteinuria, hematuria, hypertension, and slow progression of renal hypofunction accompanied by a family history. Renal biopsy is of vital importance to the diagnosis. The key pathogenesis of GFND is the deposition of FN in glomeruli. Genome sequencing technology may be helpful for the early diagnosis of GFND.


We found that changes in flowering plant composition and diversity under the N and P treatments did not alter the pollinator richness or abundance. The N and P treatments also had limited effects on the plant–pollinator interactions, including the interaction numbers, visit numbers, plant and pollinator species dissimilarity, plant–pollinator interaction dissimilarity, average number of pollinator species attracted by each plant species (vulnerability), and average number of plant species visited by each pollinator species . However, the N + P treatment increased the species and interaction dissimilarity in flowering plant and pollinator communities and decreased the generality in plant–pollinator interactions. These data highlight that changes in flowering plants caused by N + P enrichment alter pollination interactions between flowering plants and pollinators. Owing to changes in flowering plant communities, the plant–pollinator interactions could be sensitive to the changing environment in alpine regions.Soil eutrophication from atmospheric deposition and fertilization threatens biodiversity and the functioning of terrestrial ecosystems worldwide. Increases in soil nitrogen (N) and phosphorus (P) content can alter the biomass and structure of plant communities in grassland ecosystems; however, the impact of these changes on plant–pollinator interactions is not yet clear. In this study, we tested how changes in flowering plant diversity and composition due to N and P enrichment affected pollinator communities and pollination interactions. Our experiments, conducted in a Tibetan alpine grassland, included four fertilization treatments: N (10 g N m The structure and function of biodiversity, such as the biomass and diversity of plants and the animals that depend on them for survival, are vital for the sustainability of ecosystems . However2 (The availability of nitrogen (N) is considered to be the most important nutritionally limiting factor for primary productivity in grasslands . Althoug2 , dramati2 . Nutrien2 . Phospho2 . This re2 , in addi2 . Some st2 . For exaThe Tibetan grassland ecosystem covers over 60% of the Qinghai-Tibet Plateau; however, this region is experiencing increased atmospheric N deposition and local warming . These gHere, we experimentally manipulated the addition of nutrients to investigate whether and how the addition of P and N affected flowering plant communities, pollinator assemblages, and plant–pollinator interactions . In addi–1; organic carbon, 63.1 g kg–1; nitrogen, 5.75 g kg–1; and phosphorus, 0.79 g kg–1 , Qinghai, China . The res9 g kg–1 . The pla9 g kg–1 . The floruticosa , which aruticosa .4N2O) and 50 kg of triple superphosphate [Ca(H2PO4)2⋅CaHPO4] per hectare per year. Our experiment included 24 plots (4 treatments × 6 blocks) at 6 m × 6 m each , we created four transects (1 m × 5 m) within each plot in 2019. From early July to late August 2019, we counted the number of flowering plant species and flowers for each plant four times per half month along the four transects of each plot.To examine the diversity and number of pollinating insects, from early July to late August 2019, we monitored flowers for 30 min in each transect (1 m × 5 m) four times on clear days in the presence of strong wind (09: 00–19: 00). Using this observation method, each plant species was observed in each transect for 120 min. If an insect encountered the sexual components of the flowers (anthers or stigmas), we recorded a pollination visit without considering the effectiveness of the pollination . As few To estimate the completeness of the sample, we divided the observed richness by the estimated abundance-based richness estimator, Chao1, using the vegan package (v2.5-7) with the R statistical software v4.0.3, .To determine the effects of nutrient addition on the diversity and abundance of flowering plants and pollinators, we counted the flowering plant species, pollinator species, flowers, pollinator individuals, plant–pollinator unique interactions, and visits per plot.To determine whether the core generalist flowering plant and pollinator species changed with different nutrient treatments, we used UCINET (v6.0) to calculate the eigenvector centrality score  of each plant and pollinator species of different nutrient treatments . In addiTo determine the effects of nutrient addition on the dissimilarity between flowering plants and pollinators, we quantified the species and interaction dissimilarity (species or link differences in plant–pollinator interaction networks) based on the methods of To determine the impact of nutrient addition on the changes in links between plants and pollinators, we calculated two quantitative network metrics, vulnerability and generality, using the methods of The data were checked for normality and homogeneity of variance before performing the ANOVA. If the assumptions of normality and variance were not met, the data were log-transformed. We then used two-way ANOVAs in the R basic package to determine the effects of nutrient enrichment on plants, pollinators, and their interactions. The N and P treatments were fixed factors, and the block was an error term. The variables included five flowering plant and pollinator variables  and five pollination interaction variables . In addition, three other statistical analyses  were performed.First, we used a one-way ANOVA to examine the effects of the nutrient-addition treatments  on each response variable .Second, we applied the pairwise permutational multivariate analysis of variance (PERMANOVA) using the vegan package (v2.5-7)  to evaluFinally, we applied piecewise structural equation modeling in the piecewise SEM package (v2.1.2)  to clariIn total, we found that 20 flowering plant species  were visO. kansuensis, Oxytropis ochrocephala, and Oxytropis qinghaiensis). By contrast, the N + P treatment further reduced the diversity of rare species . The abundance of flowers was similar to that for the control and N or P treatments  were core generalists with the control treatment. By contrast, only two species  were core generalists with the P treatment  and the final model are qualitatively similar , which explained about 75% of the variance of the generality . The final model revealed that the change in flowering plant species caused by the N and P treatments indirectly affected the generality through changing the flower abundance and vulnerability  and increased the generality . In addition, the P treatment directly increased the abundance of flowers  and deceased the generality .The full piecewise SEM  can only change their distribution over generations, so they can become locally extinct under long-term fertilization.Soil eutrophication due to N and P addition can increase the biomass of grasses and decrease the biomass and diversity of flowering plant species, such as forb and legumes, in nutrient-limited grasslands . For exaR. membranaceus, E. regelii, and G. verum disappeared after eight years of N + P enrichment. N enrichment affects the N and P balance in the soil, changing the situation from N limitation to P limitation (D. sophia plants (D. sophia (Additionally, this research revealed that P addition further enhanced the negative effect of N enrichment on flowering plant species in alpine grasslands . For examitation . As P admitation , this comitation . For exaa plants , as N en. sophia . Further. sophia , indicatD. sophia and Angelica nitida could attract more than ten pollinator species. Additionally, our results showed that the main pollinator species were flies, bees, and butterflies (Contrary to our second hypothesis, our findings revealed that N and P addition did not alter the pollinator diversity and abundance and the core pollinator species , 3. Burkterflies , which aterflies . Therefoterflies .It is important to note that, similarly to in other studies , the scaContrary to the results of previous studies, Plant–pollinator interaction networks always exhibit nested properties and are centered around a core of generalized plant and pollinator species . Thus, cFurthermore, the addition of N or P alone did not change the core generalized flowering plants, which may not change the nestedness of plant–pollinator interaction networks, because of the generalized characteristics of pollination networks  and the We tested the importance of nutrient enrichment for changes in pollination interactions by combining network theory and pollination ecology. Our study revealed that changes in flowering plant diversity and composition, both of which are reduced by nitrogen and phosphorus enrichment, alter plant–pollinator interactions in a Tibetan alpine grassland. Future experiments in manipulating nutritional resources, and the composition and abundance of plant and pollinator species on larger temporal and spatial scales will provide important insights into how nutrient enrichment affects the response of pollinator assemblages.The original contributions presented in this study are included in the article/L-LW, Y-PY, Y-WD, and J-SH conceived the ideas and designed methodology. L-LW, FR, CZ, Z-HZ, and X-JH collected the data. L-LW analyzed the data. L-LW, Y-PY, and Y-WD led the writing of the manuscript. All authors contributed critically to the drafts and gave final approval for publication.


Background: Peyronie’s disease (PD) is a chronic inflammatory condition affecting adult males, involving the tunica albuginea of the corpora cavernosa of the penis. PD is frequently associated with penile pain, erectile dysfunction, and a secondary anxious–depressive state. The etiology of PD has not yet been completely elucidated, but local injury is generally recognized to be a triggering factor. It has also been widely proven that oxidative stress is an essential, decisive component in all inflammatory processes, whether acute or chronic. Current conservative medical treatment comprises oral substances, penile injections, and physical therapy. Aim: This article intends to show how antioxidant therapy is able to interfere with the pathogenetic mechanisms of the disease. Method: This article consists of a synthetic narrative review of the current scientific literature on antioxidant therapy for this disease. Results: The good results of the antioxidant treatment described above also prove that the doses used were adequate and the concentrations of the substances employed did not exceed the threshold at which they might have interacted negatively with the mechanisms of the redox regulation of tissue. Conclusions: We believe new, randomized, controlled studies are needed to confirm the efficacy of treatment with antioxidants. However, we consider the experiences of antioxidant treatment which can already be found in the literature useful for the clinical practice of urologists in the treatment of this chronic inflammatory disease. Peyronie’s disease (PD) is a chronic inflammatory condition affecting adult males with an autosomal dominant genetic predisposition ,2,3. TheThe disease affects the tunica albuginea of the corpora cavernosa of the penis, and in its natural evolution can also involve the tissue of the corpora cavernosa and the intercavernous septum. The disease’s hallmark is an excessive production of collagen, which causes localized fibrosis and results in penile deformation. Most of the time, the clinical consequence is a penile bend that can be more or less pronounced, and is sometimes associated with penile shortening, penile torsion of various degrees, divots, hourglass deformity, etc. The disease is frequently associated with penile pain, erectile dysfunction, and a secondary anxious–depressive state. It has been found that PD patients are more likely to have psychiatric disorders  than men without PD ,15. It iThe etiology of PD has yet to be completely elucidated, although the triggering factor is generally recognized to be local injury ,18,19. IIt is possible that older PD patients may have other associated risk factors , which further favor the progression of the disease.PD has two distinct stages: In the first (active) inflammatory stage, the inflammatory process causes the production of collagen, and the consequent remodeling of the tissue, resulting in fibrosis of the affected area (penile plaque). The plaque may then evolve towards calcification, which occurs in 20–25% of cases . The firThis article, in addition to exposing the pathogenetic mechanisms of PD, consists of a synthetic narrative review of the current scientific literature on the antioxidant therapy of this disease. The aim of this article is to show how antioxidant therapy is able to interfere with the pathogenetic mechanisms of the disease.Oxidative stress has been widely proven to be an essential component in all inflammatory processes, whether acute or chronic ,39,40,41Following a traumatic event, which need not necessarily be violent, the tunica albuginea normally reacts with tissue repair and healing. However, when trauma occurs in a male who is genetically susceptible to PD, a series of events occurs which leads to the formation of fibrotic plaque. Delamination of the tunica albuginea as a result of the injury causes an effusion of blood, with a local accumulation of fibrin that is not reabsorbed due to insufficient fibrinolysis. Fibrin accumulation is a powerful factor in the local recruitment of inflammatory cells , which immediately produce proinflammatory cytokines (interleukin-1 (IL-1), tumor necrosis factor-alpha ), and have chemotactic effects on fibroblasts . Various cells that have been attracted to the site  begin to produce important fibrogenic factors (transforming growth factor beta-1 (TGF-ß1), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF)) which induce an overproduction of collagen in situ ,52,53. A2•-) and its metabolites of hydrogen peroxide (H2O2), hydroxyl radical (HO•), hypochlorous acid (HOCl) and singlet oxygen (1O2•) [The activation of these phagocytes occurs thanks to the enzyme nicotinamide adenine dinucleotide phosphate (NADPH oxidase) present in their cytoplasm ,54,55. Tn (1O2•) ,56. A redundancy of biological signals is therefore established, producing, as a consequence, a local exacerbation of inflammation. The sources of production of the iNOS enzyme are macrophages, monocytes, T cells, smooth muscle cells, fibroblasts and myofibroblasts ,45,57. 2•)) are also reactive species such as ROS and are called reactive nitroxidative species (RNS). The production of iNOS is also stimulated by the cytokines IL-1 and TNF-alpha . The iNOThese RNS, adding themselves to ROS, cause a further oxidation state, in this case nitro-oxidation, which can cause lipid peroxidation, DNA fragmentation, the nitration of proteins, and the alteration of vascular tone (vasoconstriction) ,59. All The use of antioxidants in the treatment of PD dates back over 70 years: specifically, vitamin E was the first antioxidant to be successfully used . It was Many years later, in recent years of the second millennium, propolis began to be used—likewise, not for its known antioxidant properties, but simply because a patient with PD, who had been treated with propolis  by gastroenterologists because he was affected by Giardiasis, had noticed a significant improvement in his penile curvature. The urologists of the same hospital where the patient was treated, having observed the clinical evidence, began to treat PD patients with propolis (in oil form: propoleum). All the studies published by these Cuban urologists, which include both the exclusive use of propolis and treatment in association with laser therapy, showed significant improvement in terms of both plaque volume reduction and improvement in penile curvature ,91,92,93Subsequently, in the early 2000s, carnitine was used to treat PD, specifically for its antioxidant properties, both alone  and together with penile injections of a calcium channel blocker (Verapamil) ,95. BothA number of studies then followed in which pentoxifylline was successfully used to treat PD ,96,97,98In other studies, a number of antioxidants  were used in association with other substances  or penile traction ,99,100. Multimodal or combined treatment is a therapeutic practice which has already been used in other fields of medicine, such as oncology (polychemotherapy) and the treatment of infections (antibiotic combination therapy), for instance, in the treatment of tuberculosis or even simply in the treatment of drug-resistant bacteria, e.g., for amoxicillin-resistant germs, through the association of amoxicillin with clavulanic acid. The aim is to obtain a better therapeutic result than is achievable by administering a single substance. In our case, this is particularly advantageous, since many antioxidants also have anti-inflammatory and antifibrotic properties that differ slightly see A,B, and For instance, bilberry is capable of inhibiting the production of bFGF, unlike other antioxidant substances. Boswellia, superoxide dismutase and diclofenac cannot reduce the synthesis of PAI-1 as other substances do. Pentoxifylline, silymarin, boswellia, coenzyme Q10, carnitine, and Ginkgo biloba, unlike other antioxidant substances, are able to contrast the vasoconstriction caused by high concentrations of nitroxide radicals (NO•-), and thanks to their properties, they can cause vasodilation at the disease site ,102, thuCoenzyme Q10 is capable of activating NF-E2-related factor-2 (Nrf2) which suppresses TGF-β1 (fibrogenic factor) expression ,104. Thus, it appears that multimodal antioxidant therapy can be used as a therapeutic strategy to maximize the end effects of treatment. It must be specified that propolis is a product made by bees containing many substances that have useful properties for PD treatment: polyphenols (including flavonoids and resveratrol), cinnamic acid, caffeic acid, fatty acids, certain vitamins , and chemical elements . The most important flavonoids contained in propolis are the following: galangin, quercetin, apigenin, acacetin, catechin, chrysin, luteolin, kaempferol, pinocembrin, myricetin, naringenin, and rutin . The geoAlthough a number of guidelines contain no recommendation for the use of antioxidants in the treatment of PD, they have been used in several therapeutic experiences, either alone or in combination with other substances ,169.We searched the PubMed database for articles on the topic “antioxidant treatment in Peyronie’s disease” and found 21 articles on this topic. We considered the following articles eligible: randomized and/or controlled clinical trials; case reports containing more than one case report. After screening the 21 articles, we excluded 4 of them: 2 articles because they reported a single clinical case, and 2 clinical studies because they did not have a control group.We shall, of course, cite only a selection of the scientific literature on the topic, including randomized studies and a number of controlled studies, as well as three case report studies in which the complete regression of plaque was achieved ,175,176.Superoxide dismutase to treat PD patients. Superoxide dismutase was used as topical gel once a day for eight weeks, reaching the following statistically significant results: pain reduction in 52.6% of treated cases, compared to 20% in the control group; plaque volume reduction in 47% of treated cases, compared to the control group where, on the contrary, plaque volume grew in 8% of cases; reduction in the degree of penile curvature (between 5 and 30 degrees) in 23% of treated cases, compared to the control group in which, instead, curvature increased in 10% of cases [A randomized, double-blind, placebo-controlled study by Riedl (2005) used of cases .In a randomized controlled study by Favilla et al. (2014), the authors treated PD patients for 12 weeks with oral antioxidants  associated with weekly verapamil injections. The control group only received weekly verapamil injections . After tRegarding the other endpoints, in the group receiving oral antioxidants + verapamil injections, there were statistically significantly better results compared to the control group (only verapamil injections) in terms of: penile pain reduction, orgasmic function, intercourse satisfaction, and overall satisfaction. In conclusion, the study showed that combined therapy made it possible to obtain better results .First group: only oral therapy with pentoxifylline;Second group: only intralesional injections with verapamil ;Third group: oral pentoxifylline + intralesional injections with verapamil  .In a randomized controlled study by Alizadeh et al. (2014), the authors treated patients with PD for six months, dividing them into three treatment groups:The results were as follows: improvement in penile curvature: first group = 26.7%, second group = 36.7%, third group = 36.7%; plaque size reduction: first group = 30%, second group = 33%, third group = 33%; improvement in erectile dysfunction: first group = 46.7%, second group = 66.7%, third group = 86.7%; penile pain reduction: first group = 73.3%, second group = 76.7%, third group = 80%. Even in this study, the results show that combined therapy enables better results .–Reduction in curvature by at least 10 degrees: first group = 0%; second group = 17.8%; third group = 50%.–Improvement in the International Index of Erectile Function (IIEF) score : first group = from 17.7 to 18.5 (increase + 0.8); second group = from 20.4 to 21.6 (increase + 1.2); third group = from 20 to 22.4 (increase + 2.2).–Change in stretched penile length: first group = from 10.5 to 10.4 cm (reduction −0.1 cm); second group = from 10.7 to 10.6 cm (reduction −0.1 cm); third group = from 10.3 to 11.0 cm (increase + 0.7 cm).–Penile pain: first group = resolution of pain in 100% of cases; second group = resolution of pain in 100% of cases; third group = resolution of pain in 100% of cases .In their retrospective control group study, Gallo et al. (2019) treated PD patients for 6 months, dividing them into three groups: first group: oral therapy with arginine and pentoxifylline; second group: oral therapy with arginine and pentoxifylline + intralesional injections with verapamil ; third group: oral therapy with arginine and pentoxifylline + intralesional injections with verapamil  + penile traction therapy with a penile extender, applied daily for about 2–8 h . The theThe authors conclude that oral therapy alone can simply block disease progression, association between oral therapy and verapamil injections enables only slight improvement, and the combination of oral therapy, verapamil injections, and penile traction therapy is the only conservative approach leading to optimal results.In our 2016 retrospective control group study , we treated 206 patients with PD using the following combined therapy: group A: 112 patients, pentoxifylline  twice a month for 6 months + oral pentoxifylline + oral propolis + oral blueberry + oral vitamin E + diclofenac gel for 6 months); group B: 94 patients undertaking the same therapy as group A but with no pentoxifylline injections; group C: 101 patients with PD and no treatment .After treatment, a better response was observed in group A, where the combined therapy was bolstered by the association with pentoxifylline injections: a reduction in plaque volume in 100% of cases compared to the 79.7% obtained in group B; a mean reduction in plaque volume in 46.9% of cases compared to 24.8% of group B; an improvement in curvature in 96.8% of cases compared to 56.4% of group B; a mean reduction in the angle of the curve = −10.1 degrees compared to −4.8 degrees in group B; recovery of normal penile rigidity in patients with ED was obtained in 56% of cases compared to 23.5% of group B .In 2013, we published a controlled study  which, in contrast with other articles in the literature, proved that oral vitamin E, associated with other substances  + topical diclofenac  for 6 months, was statistically effective in curing PD. In particular, vitamin E was shown to improve the therapeutic result: increasing the reduction in the volume of plaque after treatment (from 35.8% to 50.2%); increasing the percentage of patients who achieved an improvement in their penile curvature after treatment (from 48.4% to 96.6%); and increasing the degree of reduction in the curve (from 6.7 degrees to 12.2 degrees) .Finally, we cite three case report articles of ours in which, overall, thanks to multimodal antioxidant treatment, we obtained the complete regression of plaque in eight patients suffering from PD ,175,176.In all patients, we used most of the antioxidants mentioned above . In two cases, the combined oral and topical therapy was not associated with pentoxifylline injection therapy because the patients did not give their consent.3, while the patient whose treatment took 53 months had an initial plaque volume measuring 733 mm3 and received no penile injections because of his refusal to undergo injection therapy.Treatment duration varied from 28 months to 53 months, most likely depending on plaque volume, since the patient who was cured in 28 months initially had plaque which was measured by ultrasound to be 122 mmThe time period required for treatment was necessarily long due to the nature of the disease, i.e., its being a chronic (not an acute) inflammation, which therefore requires adequate treatment time. All patients, in any case, were informed before starting treatment that treatment would have to be long, due to the intrinsic characteristics of PD.In all cases, at the end of treatment, patients experienced the complete regression of both their baseline penile pain and the penile curvature caused by the disease.Oxidative stress evidently plays a leading role in Peyronie’s disease, a disease where there is an evident redox imbalance caused by the production of reactive species, following trauma to the penis in a genetically predisposed male.It is also evident that oxidative stress plays a very large role not only in the onset, but especially in the progression of PD, resulting in local tissue damage and the formation of fibrotic plaque and its possible calcification ,21,23.From the literature, it is therefore clear that the most important physiopathogenic events are the following: penile injury (including minor trauma), formation of blood collection in the corpus cavernosum, recruitment of inflammatory cells, release of ROS and cytokines, activation of transcription factor NF-kB, the production of iNOS and the subsequent overproduction of radical NO, and the overproduction and local deposition of collagen (plaque formation) . All theThe good results of the antioxidant treatment described above also prove that the doses used were adequate and the concentrations of the substances employed did not exceed the threshold beyond which they might have instead interacted negatively with the mechanisms of redox regulation of the tissues (see “antioxidant paradox”) ,181. It Several antioxidant substances, some of which we use to treat PD, have been successfully used in other PD-related diseases. Silymarin, when given in a dose of 140 mg thrice daily for 3 months as an adjuvant for glycemic control, lipid profile, and insulin resistance, proved to have a beneficial efficacy . AdditioIn our clinical practice, in particular in the treatment of PD, we never doubted that our antioxidant doses were adequate and not excessive, especially since in our first experiences, we used at most three or four substances  ,177. OveWe believe that many current articles on the conservative treatment of PD are lacking, and unable to accurately evaluate the volume of plaque before and after the treatment. The plaque volume must be evaluated only with a very sensitive ultrasound system and a three-dimensional evaluation with the formula of the ellipsoid (volume = 0.524 × length × width × thickness). The change in plaque volume is an important endpoint that should always be evaluated, together with the other parameters, for a correct evaluation of the therapeutic result. We propose that all future studies concerning the conservative treatment of PD include this very important parameter.Oxidative stress represents the fundamental chemical environment for the onset and progression of Peyronie’s disease, and—more broadly—for chronic inflammatory states, degenerative diseases, and malignant neoplasms. We think the good clinical results obtained using antioxidant therapy confirm the important role played by oxidative stress in PD. We also believe that the experiences of antioxidant treatments already present in the literature represent an important aid to the clinical practice of urologists for the treatment of this chronic inflammatory disease which, incidentally, is not rare, as its prevalence, especially in the Western world, is similar to that of diabetes mellitus (=10.5% worldwide) .Treating PD with antioxidants directly interferes with the most important mechanisms of inflammation, thus treating the disease directly and not just its symptoms. We therefore hold that PD is a disease which can be cured, in contrast to the affirmation of authors who consider it an incurable condition . We advoWe recommend that only patients with active (unstabilized) PD be treated with medical therapy. In the case of large penile plaques, we recommend adding periodic perilesional injections with pentoxifylline to the oral antioxidant substances.In any case, we believe further studies are needed to both improve the understanding of the pathophysiology of Peyronie’s disease and to implement new randomized controlled trials to confirm the efficacy of treatment with antioxidants. We also propose multi-center studies involving the combined use of antioxidants in order to demonstrate more clearly that PD can be cured.


In particular, the central carbon metabolites (CCM) appear to be relevant for musculoskeletal regeneration, given their role in energy metabolism. However, the sensitivity, reproducibility, and robustness of these methods for detecting targeted polar CCM remains unknown. Overall, the extraction of metabolites using the mBD, mBD-low, and mMat methods appears sufficiently robust and reproducible for bone, with the mBD method slightly bettering the mBD-low and mMat methods. Furthermore, mBD, mBD-low, and mMat were sufficiently sensitive in detecting polar metabolites extracted from mouse muscle; however, they lacked repeatability. This study highlights the need for a re-thinking, towards a tissue-specific optimization of methods for metabolite extractions, ensuring sufficient sensitivity, repeatability, and robustness.The identification of endogenous metabolites has great potential for understanding the underlying tissue processes occurring in either a homeostatic or a diseased state. The application of gas chromatography-mass spectrometry (GC-MS)-based metabolomics on musculoskeletal tissue samples has gained traction. However, limited comparison studies exist evaluating the sensitivity, reproducibility, and robustness of the various existing extraction protocols for musculoskeletal tissues. Here, we evaluated polar metabolite extraction from bone and muscle of mouse origin. The extraction methods compared were (1) modified Bligh–Dyer (mBD), (2) low chloroform (CHCl Recently, there has been increasing interest in applying metabolomics to understand the pathophysiology of musculoskeletal disorders and the subsequent regeneration ,2. This To date, the sampling for metabolomics in many clinical indications such as musculoskeletal regeneration has predominantly focused on peripheral body fluids, such as serum, plasma, and urine ,4. Such Consequently, the quantification of metabolites using mass spectrometry (MS)-based metabolomics often results in more heterogeneity and variability for tissue samples, i.e., bone or muscle, than peripheral body fluids . Therefo3) was varied, and the modified Matyash (mMat) method, where CHCl3 was substituted with Methyl tert-butyl ether (MTBE), a chemical with better stability and less toxicity than CHCl3. Subsequently, efforts were aimed at comparing the repeatability and sensitivity of the methods, using gas chromatography-mass spectrometry to detect the CCM.Following tissue homogenization, metabolite extraction is a crucial step, mandating the reproducibility of results and the possible range of metabolites that can be detected. Generally, the choice of metabolite extraction depends on the analytical tools and the metabolites of interest. This study focused on the central carbon metabolites (CCM), the collection of products that result from the complex networks of reactions responsible for the transport and oxidation of the main sugars inside a cell. This include metabolic pathways such as the tricarboxylic acid cycle, pentose phosphate pathway, amino acids, glycolysis, etc. . For theThe performance of the methods was compared by targeted analysis of 75 specific metabolites from the CCM. The following biological classes were covered: glycolysis, amino acids and metabolites from the tricarboxylic cycle, as well as some chemical classes, herein named as “others” , as already demonstrated in previous studies ,14.Data reproducibility is usually reported as the relative standard deviation (RSD in %) of a repeated sample measurement in the metabolomics field. In the GC-MS metabolomics analysis, the maximum generally accepted tolerance of RSD is 30% for any individual metabolite . An RSD Physical disruption of the tissue is a prerequisite for successful metabolite extraction , especiaBone samples processed using the Tissuelyzer yield an mRSD value of 31% , varyingThis study used methoximation (MeOx) followed by trimethylsilylation (TMS) to derivatize the samples. Such derivatization can yield incomplete or isomeric derivative peaks. The repeatability of derivatives of TMS and MeOX products are listed in n = 5) were homogenized using the Tissuelyzer, and the polar metabolites were extracted following the mBD, mBD-low, or mMat method. All samples were analyzed as a single batch on the GC-MS. The three extraction methods were compared in terms of sensitivity (the number and intensity of metabolites detected), robustness , and repeatability (RSD distribution and mRSD). Although five replicates per class were initially prepared , four samples were lost due to missed injections, leaving four per class for mBD and mBD-low, and three per class for mMat.The mice bones  to 52% (D-pantothenic acid) for individual compounds b. Among Using the mBD-low method, the mRSD for all the 60 metabolites detected was 18, ranging from 3% (myoinositol) to 79% (ribose-5-phosphate) for individual compounds. Of which, 15 metabolites had RSDs of less than 10%, 29 metabolites with RSD between 10 and 30%, and 16 metabolites with RSDs >30%.Metabolites extracted using the mMat method had a mRSD of 15%, with RSDs of individual compounds ranging from 1%  to 77% (pyruvic acid). Of the 59 metabolites detected, 24 metabolites showed RSDs of less than 10%, 27 metabolites showed RSDs between 10 and 30%, and eight metabolites had RSDs >30%.The repeatability (as measured by the mRSD) of the metabolites based on biological classes was best for tricarboxylic acid cycle , followed by glycolysis  and amino acids .p = 0.016. The null-hypothesis was then rejected, and the number of metabolites detected between the three methods was considered significantly different.The three methods were relatively comparable in terms of robustness, as the number of detected and missing metabolites were similar for all biological repeats, as illustrated in Sensitivity was additionally assessed by comparing the relative intensities of individual metabolites across the three methods . For thin = 5) were processed following the homogenization and polar metabolites extraction methods previously used for mouse bone. As illustrated in the Venn diagram  to 112% (erythrose-4-phosphate) for individual compounds. Among the 63 detected metabolites, two metabolites had an RSD of less than 10%, indicating very good reproducibility, 25 metabolites had RSDs between 10% and 30%, and 36 metabolites had RSDs >30%.Using the mBD-low method gave an mRSD of 46% across the 59 metabolites detected, ranging from 7% (adenosine and lactic acid) to 88% (pyruvic acid) for individual compounds. Of which, two metabolites had RSDs of less than 10%, 17 metabolites had RSDs between 10% and 30%, and 40 metabolites had RSDs >30% b. Metabolites extracted from mouse muscle using the mMat method yielded an mRSD of 47%, with RSDs of individual compounds ranging from 8% (pyruvic acid) to 93% (ribitol). Of the 57 metabolites detected, two metabolites showed RSDs of less than 10%, 15 metabolites showed RSDs between 10 and 30%, and 40 metabolites had RSDs >30%. The repeatability (mRSD) of the metabolites based on biological classes, i.e., glycolysis  and amino acids , were above 30% for all extraction methods. The mRSD of metabolites of the tricarboxylic acid cycle  was only marginally lower than that of metabolites from the glycolysis or amino acids families.The three methods were relatively comparable in terms of robustness, as the number of detected and missing metabolites was similar for all biological repeats, as illustrated in The sensitivity for individual metabolites is illustrated in Metabolomics is a powerful platform for high-throughput quantification of small molecule metabolites of biological specimens. Prior to the screening with GC-MS, acquired tissue specimens, i.e., bone or muscle, must undergo tissue homogenization, metabolite extraction, and derivatization. However, there is a lack of consensus on the optimum methods for extracting metabolites from tissue specimens, especially if different tissues such as bone and muscle are compared. The first problem to overcome with solid tissues, such as bone and muscle, is finding a way to measure technical replicates without distorting the extraction process. Here, biological replicates from in-bred healthy mice were used, with the assumption that the within-group biological differences between the mice would be less than the between-method technical differences between the extraction methods. Moreover, in this study, tissue specimens were harvested from male mice, thereby minimizing the biological variability by mitigating the influence of sex on the metabolome, as described by Caterino et al. . NonetheHomogenization of tissue is also a crucial step that allows for sufficient penetration of solvents through the tissue matrix to extract metabolites. Generally, homogenization is more challenging for hard tissue, i.e., bone, than soft tissue, i.e., muscle. A previous study indicated that the 24 Precellys homogenizers were optimal for the homogenization of muscle . Here, tThe suitability of the three extraction methods  for extracting metabolites from mouse bone and muscle was assessed in terms of sensitivity, robustness, and repeatability for targeted CCM profiling. Extraction methods were based on published literature ,14,15. A3 present in mBD compared to mBD-low, enabling full extraction of the lipids into the lipid phase and resulting in better detection of polar metabolites, with less contamination of non-polar compounds. Similarly, Nam et al. trifluoroacetamide (MSTFA) was added to the solution, incubated at 37 °C for 60 min and centrifuged for 10 min at 18,213× An identification mixture was prepared and derivatized using the same method, to ensure reliable compound identification. Additionally, an alkane mixture for a reliable retention index calculation was included . Pooled The GC-MS analysis of CCM was performed on a Pegasus 4D GCxGC TOFMS-System  complemented with an auto-sampler . The samples were injected in split mode  in a temperature-controlled injector with a baffled glass liner . The following temperature program was applied during sample injection: 2 min to allow for column equilibration at 68 °C, followed by a serial increment of temperature, from 68 °C to 120 °C (5 °C/minute), from 120 °C to 200 °C (7 °C/minute), and from 200 °C to 320 °C (12°C/minute). The maximum temperature, 320 °C, was maintained for 7.5 min. m/z with 10 spectra/second. Gas chromatographic separation was performed in one-dimensional mode on an Agilent 7890 , equipped with a VF-5ms column  of 30 m length, 250 µm inner diameter, and 0.25 µm film thickness. Helium was used as the carrier gas, with a flow rate of 1.2 mL/minutes. Spectra were recorded in a mass range of 60 to 600 All samples were run in scan mode, to obtain targeted and untargeted metabolic profiles. We pre-selected 75 key CCM to target for data analysis , based oThe GC-MS chromatograms were processed with ChromaTOF software  including baseline assessment, peak picking, and area computation.Once the pre-processing of GC-MS data was completed, the data were exported and merged with an in-house written R script. A total of 5 biological replicates were prepared for each method. A minimum of 3 valid values per metabolite had to be present to consider the metabolite as present. The peak area of each metabolite was calculated by normalization to the internal standard, cinnamic acid. Relative quantities were used, and derivatives from the same original metabolite species were summed up for the final data analysis. A Kruskal–Wallis test was performed using IBM SPSS Statistics 27.0 for statistical analysis. R studio (Version 1.3.1056) software was used for data analysis purposes. The analyzed metrics included the number of compounds, missing values, and the mRSDs of individual metabolite and biological classes.Generally, this study highlights the relevance of the careful preparation of tissue sampling and processing, specifically if such diverse tissues as bone and muscle are to be processed before GC-MS. In the first step of tissue processing, the results indicated that Tissuelyzer yielded a better homogenate of mouse bone for extracting polar metabolites than Pulverizer. While extracting polar metabolites from bone or muscle, the types and ratio of the solvent mixture and the tissue to solvent ratio can impact the sensitivity, robustness, and reproducibility of the GC-MS profiling of CCM.