CXCL12, SSTR4, FABP5, SLC2A1, MYLK4, and EIF4E3. KEGG pathway analysis identified that some DEGs were significantly enriched in the calcium signaling pathway, which may promote muscle growth. The gene–gene interaction network of DEGs was mainly related to the transmission of cell signals and substances, hematological system development, and functions. This study can provide theoretical guidance for the production and breeding management of the Shitou goose and Wuzong goose and help reveal the genetic mechanisms underlying diverse body sizes between two goose breeds.Goose is an important poultry commonly raised for meat. The early growth performance of geese significantly influences their market weight and slaughter weight, affecting the poultry industry’s economic benefits. To identify the growth surge between the Shitou goose and the Wuzong goose, we collected the early growth body traits from 0 to 12 weeks. In addition, we investigated the transcriptomic changes in leg muscles at the high growth speed period to reveal the difference between the two geese breeds. We also estimated the growth curve parameters under three models, including the logistic, von Bertalanffy, and Gompertz models. The results showed that except for body length and keel length, the best-fitting model between the body weight and body size of the Shitou and Wuzong was the logistic model. The growth turning points of Shitou and Wuzong were 5.954 and 4.944 weeks, respectively, and the turning point of their body weight was 1459.01 g and 478.54 g, respectively. Growth surge occurred at 2–9 weeks in Shitou goose and at 1–7 weeks in Wuzong goose. The body size traits of the Shitou goose and Wuzong goose showed a trend of rapid growth in the early stage and slow growth in the later stage, and the Shitou goose growth was higher than the Wuzong goose. For transcriptome sequencing, a total of 87 differentially expressed genes (DEGs) were identified with a fold change ≥ 2 and a false discovery rate < 0.05. Many DEGs have a potential function for growth, such as  China is the largest goose producer and market for meat geese in the world. In 2021, 570 million commercial geese were sold, and the output value of meat geese is 52.26 billion yuan, 6.8% higher than that in 2020 [Geese have the fastest initial growth rate of poultry , and theGrowth traits are critical economic traits in the poultry industry that are controlled by multiple genes and the environment . In receThe Shitou goose is the largest goose species in the world, with many advantages, such as fast growth, enormous weight, and affluent muscle . The aveIn this study, the body weight and body size traits of 0–12 week-old Shitou and Wuzong geese were measured, and the growth curves of the body weight and body size traits were fitted and analyzed using the logistic, von Bertalanffy, and Gompertz models. In order to explore the genetic mechanisms underlying goose growth performance, we applied comparative transcriptome analysis of large-size Shitou goose with small-size Wuzong goose at the age of peak growth. The results of this study are useful for understanding the mechanisms regulating the development of leg muscle and the pattern of goose growth.All experimental animal procedures complied with the laboratory animal management and welfare regulations approved by the Experimental Animal Committee of Zhongkai University of Agriculture and Engineering, Guangzhou, Guangdong, China. This study was approved by the Experimental Animal Committee of Zhongkai University of Agriculture and Engineering (NO.2019110220). All efforts were made to minimize animal suffering.The largest-size Shitou goose and the smallest-size Wuzong goose from Qingyuan Jinyufeng Goose Industry Co., Ltd.  were used in this experiment. Forty one-day-old Shitou geese and forty one-day-old Wuzong geese were randomly selected and fed to twelve weeks of age. All animals used in this experiment were raised in free-range under the same conditions with a free diet and supplemented with green grass. Three male Shitou geese and three male Wuzong geese were selected and slaughtered at 5 weeks of age. After slaughtering, leg muscle was collected for transcriptome sequencing and real-time PCR validation.The measurement of body weight and body size traits refers to the statistical method for measuring poultry production performance issued by the Ministry of Agriculture of China . The measurement items include body weight, body length, chest depth, chest width, pelvis width, keel length, tibia length, shank circumference, semi-submersible length (waterfowl), and neck length.The SPSS 17.0 software package  was used to analyze the data. Three nonlinear growth models, including logistic, Gompertz, and von Bertalanffy, were used to fit the growth curves of the geese ,6,7. TheAccording to the manufacturer’s instructions, total RNA was extracted from the leg muscle tissues with Trizol reagent . RNA purity was checked with the RNeasy Animal Mini Kit , and RNA quality and concentration were checked with Agilent 2100 BioAnalyzer  and QUBIT RNA ASSAYKIT (Invitrogen). Subsequently, genomic DNA was removed by using RNase-Free DNase I (Qiagen). The total RNA of each individual was used to construct a cDNA library for RNA-seq. Six cDNA libraries were prepared and were sequenced through the Illumina HiSeq2000 system .http://www.ingenuity.com, accessed on 1 September 2019).Raw reads were filtered by removing adaptors and low-quality reads with Trim Galore. Subsequently, the clean reads were mapped to the reference genome   using To−ΔΔCt method with GAPDH as the control [To verify the reliability of the RNA sequencing data, 11 DEGs were randomly selected for qRT-PCR validation. The primers were designed using Primer Premier 5.0 software . qRT-PCR control . The rea2 higher than 0.96  and the Wuzong goose . The clean data from each sample reached more than 6.66 Gb. The percentage of Q30 base in each sample was greater than 92.36%, and the GC content reached 50.87–52.02% . The comA total of 87 DEGs  were ideST8SIA6, B3GAT2, SSTR4, and LOC106030438, were enriched for GO terms involved in the growth and development of muscle.GO enrichment analysis was used to annotate the function of DEGs. The GO enrichment includes three categories: biological process, cellular component, and molecular function . In termPathway enrichment analysis was performed to gain insight into the functional roles of DEGs in the leg muscle between the two goose species. Four significantly enriched pathways were identified, including the metabolism of xenobiotics by cytochrome P450, glutathione metabolism, the calcium signaling pathway, and vascular smooth muscle contraction. There were seven DEGs involved in the four pathways .ADORA2A, COL4A5, CXCL12, FABP5, PCDH19, and SLC2A1, and 10 downregulated genes, including ANKRD1, EZR, HSPB1, MAP3K4, NOP16, ODC1, P38 MAPK, RPS15, RSAD2, and THBD , as a signal molecule, is mediated by the family of SST receptors (SSTRs) on the cell membrane and plays an important role in inducing cell apoptosis, inhibiting tumor cell proliferation, inhibiting the function of insulin, and inhibiting cell growth and other biological processes ,27,28, sCXCL12 is crucial for promoting developmental myogenesis and plays an essential role in muscle growth and angiogenesis in skeletal muscle [CXCL12 was recognized to play a major role in the maintenance, development, and differentiation of progenitor stem cells in the musculoskeletal system [CXCL12 in the Shitou goose was higher than that in the Wuzong goose, which may promote muscle development and growth in the Shitou goose.l muscle ,30. CXCLl system . In thiseIF4E), which is considered the cornerstone in the cap-dependent translation initiation machinery, is implicated in cell transformation, tumorigenesis, and angiogenesis by facilitating the translation of oncogenic mRNAs [eIF4E3, whose function is dependent on its atypical cap-binding activity, is an inhibitor rather than a promoter of both target transcript expression and oncogenic transformation [eIF4E3 in the Wuzong goose was higher than that in the Shitou goose, which may inhibit growth in the Wuzong goose.Eukaryotic translation initiation factor 4E  encoded by the SLC2A1 gene mediate the facilitated transport of glucose, which is the primary energy substance of tissue cells, and the striated skeletal muscle in birds is a central glucose-utilizing tissue. Therefore, the SLC2A1 gene can induce skeletal regeneration [Three critical gene networks were identified through the analysis of the gene interaction network. The first network is related to cell-to-cell signaling and interaction, cellular assembly and organization, and hematological system development and function, in which neration ; at the neration . GLUT-1 FABP5 is the delivery of ligands to PPARd, which promotes cell migration, proliferation, and survival [FABP5 can promote cell growth and proliferation through the transmission of cell signals and substances, which also promotes the growth and development of the body.The function of FABP is to store and transport fatty acids in cells for a short time and participate in the transport and metabolism of fatty acids. It can transport fatty acids from the cell membrane to the sites of fatty acid oxidation, triglyceride, and phospholipid synthesis . FABP5 isurvival ,49,50. TCXCL12, SSTR4, FABP5, SLC2A1, MYLK4, and EIF4E3. KEGG pathway analysis identified that some DEGs significantly enriched in the calcium signaling pathway, which may promote muscle growth. The interaction network of DEGs was mainly related to the transmission of cell signals and substances, hematological system development, and function. The results of this study will help understand the genetic mechanisms of goose growth and development.In this study, we determined the time of rapid growth between Shitou geese and the Wuzhong geese. We identified 87 DEGs by RNA-seq, some which have a potential function on growth, such as


To investigate the clinical features of active tuberculosis (TB) infection due to immune checkpoint inhibitors (ICIs) treatment in patients with advanced cancer.We report the diagnosis and treatment of a case of pulmonary malignancy , secondary to active TB infection following ICIs therapy. Moreover, we summarize and analyze other related cases collected from the China National Knowledge Infrastructure (CNKI), Wanfang Database, PubMed, the Web of Science, and EMBASE (up to October 2021).A total of 23 patients, including 20 males and 3 females who were aged 49-87 years with a median age of 65 years, were included in the study. Twenty-two patients were diagnosed by Mycobacterium tuberculosis culture or DNA polymerase chain reaction (PCR), while the remaining patient was diagnosed by tuberculin purified protein derivative and pleural biopsy. One case had an interferon-gamma release assay (IGRA) to rule out latent TB infection prior to the application of ICI. Fifteen patients received an anti-tuberculosis regimen. Among the 20 patients with a description of clinical regression, 13 improved and 7 died. Seven of the patients who improved were treated with ICI again and four of them did not experience a recurrence or worsening of TB. The case diagnosed in our hospital also improved after receiving anti-TB treatment after stopping ICI therapy, and continued chemotherapy on the basis of anti-TB treatment, and his condition is relatively stable at present.Due to the lack of specificity of TB infection following ICIs therapy, patients should be followed for fever and respiratory symptoms for 6.3 months after drug administration. It is recommended that IGRA should be performed before ICIs therapy and the development of TB during immunotherapy in patients who are positive in IGRA should be closely monitored. The symptoms of TB in most patients can be improved with ICIs withdrawal and anti-TB treatment, but there is still a need to be alert to the potentially fatal risk of TB. SomeImmunotherapy represents an important and growing area of clinical oncology. Immune checkpoint inhibitors (ICIs) exert their antitumor activity by inhibiting the suppression pathway of immune cells, which can enhance the activity of immune cells and reduce T cell depletion . CurrentIn the present study, a case of active TB associated with the application of camrelizumab in 2022 was studied and a systematic review of the collected related literature was taken to further elucidate the potential impact of ICIs on the development of active TB.22.1One patient with active TB infection following camrelizumab treatment who was admitted to Shengli Oilfield Central Hospital and patients collected through literature search with TB associated with ICIs.2.2We first consulted and organized the electronic medical records of the patient admitted to Shengli Oilfield Central Hospital to form a case report. Moreover, we searched through the China National Knowledge Infrastructure (CNKI) and Wanfang Database with the terms ‘ICIs’ and ‘TB’ up to October 2021, but no similar reports were found in China. Then, the compound terms ‘TB’ and ‘PD-1’ or ‘PD-L1’ or ‘CTLA-4’ or ‘pembrolizumab’ or ‘nivolumab’ or ‘caliplimab’ or ‘atezolizumab’ or ‘avelumab’ or ‘duvalumab’ or ‘ipilimumab’ or ‘temlimumab’ were searched for on PubMed and the Web of Science. Furthermore, ‘TB’ and  were searched for on EMBASE. Each of the obtained results was relevant to the topic of TB in ICIs therapy, and the data provided in the cases were relatively complete.In addition, an Excel data extraction sheet was applied to extract relevant data on patients from clinical records and literature, including general data , ICIs application, applications of other immunosuppressants, the development of TB (time lag from ICIs administration to TB occurrence and laboratory results of TB), treatment and regression of TB, and the reapplication of ICIs. Finally, we performed statistical analysis of the collected data.33.1The patient, a 68-year-old male, was admitted to our hospital because of “cough and sputum with breathlessness for 3 months”. The patient presented with cough and sputum 3 months ago. Specifically, the patient had a paroxysmal cough with white mucous sputum, accompanied by chest distress, shortness of breath, and numbness of both lower limbs. He had a previous history of smoking for 50 years (20 cigarettes/day) but had quit smoking for 5 months. The rest of his past medical history was negative.Physical examination: general condition was acceptable, superficial lymph nodes were not palpably enlarged, and the breath sounds of both lungs were rough without rhonchus and moist rales. No abnormalities were found on cardiac or abdominal examination.The examinations undertaken are listed in 1. Chest CT plain scan and enhanced scan: : abnormal lesion signal shadow was found in the anterior horn of the left lateral ventricle, which was presumed to be a cavernous hemangioma.3. Tracheoscopy (09 March 2022): cauliflower-like neoplasm was found to block the orifice in the upper lobe of the left lung, and the opening of the lower lobe was slightly compressed and narrowed; no abnormalities were found in the bronchial tubes of the bilateral lobes.4. Tumor markers (03 March 2022): carcinoembryonic antigen (CEA) was 6mg/ml, CA125 was 88.18U/ml, section 19 of cytokeratin was 12.91ng/ml, and neuron-specific enolase (NSE) was 33.72ng/ml.There were no abnormalities in the routine blood test, whole-body bone imaging, biochemistry analysis, blood clotting, echocardiography, and electrocardiogram.M. tuberculosis, Pneumocystis carinii, Actinomyces, nocardiosis, and cytomegalovirus.Pathological findings: the moderately differentiated squamous cell carcinoma was revealed (through bronchoscopy biopsy of left upper lobe lung). Immunohistochemical findings: CK5/6 (+), CK7 (-), Ki-67 , NapsinA (-), P40 (+), TTF-1 (-), and P63 (+). No abnormalities were found in etiological examinations for bacteria, fungus, Final diagnosis: there was a malignant tumor  with obstructive pneumonia in the upper lobe of the left lung. Since the patient had no surgical indications, he was suggested for chemotherapy combined with radiotherapy and PD-L1 testing. Genetic testing showed negative results for EGFR, ALK, ROS1, and PD-L1 expression.Treatment:9/L and neutrophils dropping to 0.23 × 109/L. Also, the patient was given symptomatic treatment such as leuke-raising injections but refused to accept another cycle of chemotherapy. After excluding contraindications, he was treated with 200 mg of camrelizumab for immunotherapy on 25 April 2022. On 21 May 2022, the patient began to cough up blood in phlegm with a volume of about 20 ml and he was reviewed using a chest CT on 22 May 2022 , two cases of oral squamous cell carcinoma, one case of nasopharyngeal carcinoma, one case of Hodgkin lymphoma, and one case of Merkel cell carcinoma.3.3All patients were treated with PD-1 inhibitors. Only one patient  received3.4Only one patient  screenedTwo of the 23 patients were presumed to have latent TB before the application of ICIs. One of them  was from3.5Twenty-two of the 23 cases were confirmed to have TB by the culture of Mycobacterium TB or by PCR analysis of TB DNA. The analytical specimens were mainly sputum or bronchoalveolar lavage fluid, but reactivation of TB in other organs  was also recorded. The remaining case  was diag3.6Immunotherapy was discontinued in all but five patients , 18, 19 The specific anti-TB regimens of eight patients were not listed. Of the other 15 patients (including the patient in the study), 11 of them were initially treated with isoniazid, rifampicin, pyrazinamide, and ethambutol , 21, 22,Seven patients died during follow-up. The first  died int4Our results suggest that inhibition of the PD-1/PD-L1 pathway is associated with the development of TB, and the ICIs leading to TB was a PD-1 inhibitor in all cases. Moreover, there were no cases of TB disease found that occurred during treatment with CTLA-4 inhibitors. In the majority of cases, TB-related signs and symptoms improved with TB treatment, but two patients died of TB-related complications. Moreover, our findings also suggest that active TB can develop in a variety of organs other than the lungs.It has been demonstrated that the PD-1/PD-L1 pathway is closely related to the pathophysiology of TB. PD-1 (CD279/PDCD1) is a cell surface inhibitory receptor that is expressed on activated T and B cells upon binding to the Ag receptor. PD-1 binds to either of its two ligands, PD-L1 (B7-H1/CD274) and PD-L2 (B7-DC/CD273), inhibiting T cell proliferation and cytokine secretion. ICIs can relieve the immunosuppression of T cell activation by tumor cells, and promote the activation and proliferation of T cells, thereby killing tumor cells. When the body is infected with TB, MTB is phagocytosed by macrophages and presented to T cells after processing by antigen-presenting cells, and then T lymphocytes secrete a variety of cytokines, such as γ interferon (IFN-γ) and tumor necrosis factor (TNF)-α, to activate the anti-TB activity of macrophages. When TB granulomas are formed, the body presents latent tuberculosis infection, at which the expression of PD-1 increases, inducing apoptosis of T cells. ICIs can block PD-1, restore the function of lymphocytes, release inflammatory cytokines such as IFN-γand TNF-αin T cells again, and excessive inflammatory cells and cytokines destroy the extracellular matrix, which is conducive to the growth of MTB, disrupts the homeostatic control of latent pulmonary tuberculosis infection, and active tuberculosis infection occurs , 18. FurTB screening in high-risk patient groups, particularly those exposed to epidemiological risk factors for TB, should be applied to patients before the initiation of immunotherapy. In this study, 10 of the 19 patients were from East or South East Asia where TB is endemic. If patients receiving anti-PD1 antibodies in areas with high TB prevalence develop fever and respiratory symptoms, the reactivation of TB should be considered after respiratory infection and pneumonia has been excluded. Therefore, latent TB screening needs to be applied before starting ICIs therapy in TB-endemic countries. A previous study has shown that adequate treatment of latent TB infection significantly reduces the risk of active TB in these patients . AlthougAlthough acute TB infection is diagnosed according to clinical symptoms, chest X-ray, and sputum culture or PCR, the initial symptoms and radiological findings may not distinguish between tumor progression, TB infection, or changes in immune-mediated imaging. Then, IGRA testing should be performed before the treatment of immune checkpoint inhibitors therapy to rule out the possibility of latent Mycobacterium TB infection. Only one of these patients took IGRA testing before ICIs administration. The IGRA result of the patient, which was negative at first, changed to positive after new abnormalities on imaging, which contributed to the diagnosis of TB. Therefore, it is necessary to take IGRA testing before the treatment of immune checkpoint inhibitors therapy.It is an important clinical question whether ICIs therapy can be continued after the development of active TB. Given that continued treatment with ICIs may worsen TB, it is reasonable to discontinue ICIs after the diagnosis of active TB. However, the appropriate time to restart ICIs therapy is still being worked out. Although the four reported patients who restarted immunotherapy did not have a relapse or worsening of TB, these four cases are not sufficient to confirm the appropriateness of restarting ICIs therapy in the presence of TB. In clinical practice, the decision to continue, temporarily interrupt, or permanently suspend treatment with ICIs should take several aspects into account, including the severity of MTB infection, tumor control, and the severity of complications. Therefore, it is better to let multidisciplinary specialists in oncology discuss whether ICIs can be restarted or not.Above all, ICIs have revolutionized cancer treatment, namely, greatly improving the overall survival of patients. However, potential MTB infection or reactivation of the disease during the treatment with ICIs still needs to be considered. It is recommended that IGRA testing be performed before ICIs therapy, and the development of TB during immunotherapy should be closely monitored for in patients who are positive in the results of IGRA testing to enable early diagnosis and timely treatment.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Shengli Oilfield Central Hospital. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article. The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Institutional Review Board of Shengli Oilfield Central Hospital (No. 2022026). All the study subjects provided informed consent.SL, CL, and GX designed the overall research strategy. CL collected the clinical information. CL and GX wrote the manuscript. CL, GX, TF, and SG participated in data discussion. All authors contributed to the article and approved the submitted version. CL and GX contributed equally to this work and share first authorship.


The purpose of this study was to investigate the relationship between bedtime at night and the risk of diabetes in adults.We extracted data from 14,821 target subjects from the NHANES database for a cross-sectional study. The data on bedtime came from the question in the sleep questionnaire: “What time do you usually fall asleep on weekdays or workdays?”. Diabetes was defined as fasting blood sugar ≥ 126mg/dL, or glycohemoglobin ≥ 6.5%, or 2-hour Oral Glucose Tolerance Test blood sugar ≥ 200mg/dL, or taking hypoglycemic agent and insulin, or self-reported diabetes mellitus. A weighted multivariate logistic regression analysis was conducted to explore the relationship between bedtime at night and diabetes in adults.From 19:00 to 23:00, a significantly negative association can be found between bedtime and diabetes. From 23:00 to 02:00, The relationship between the two was positive, nevertheless, the P values was not statistically significant(p = 0.3524). In subgroup analysis, from 19:00–23:00, the relationship was negative across genders, and in males, the P-values were still statistically significant(p = 0.0414). From 23:00–02:00, the relationship was positive across genders.Earlier bedtime (before 23:00) increased the risk of developing diabetes. And this effect was not significantly different between male and female. For bedtime between 23:00–2:00, there was a trend of increasing the risk of diabetes as the bedtime was delayed. Type 2 diabetes is a chronic disease whose incidence is increasing at an alarming rate around the world. In the early 21st century, the International Diabetes Federation (IDF) estimated that there were 151 million adults with type 2 diabetes worldwide , physicaSleep factors have been illuminated to be associated with the development of diabetes in many cross-sectional and prospective studies. A meta-analysis (prospective research) involving 482,502 participants showed a U-shaped relationship between sleep duration and diabetes, with 7–8 hours of sleep per day having the lowest risk of developing diabetes . AnotherThe NHANES database is a national health and nutrition survey of the U.S. population that collects data on a biennial basis. Since 2015, the database has collected data on bedtime in sleep questionnaires. So we intend to extract relevant data from the database to study the effect of bedtime on diabetes.Fig 1).Data from 2015-March 2020 pre-pandemic were extracted for analysis. The total sample size is 25,531 subjects, and the final target population is 14,821. Participants will be excluded if they:1) missing data on the diagnosis of diabetes(n = 970); 2) missing bedtime data and bedtime between 8:00 and 18:00(n = 8522); 3) under the age of 18(n = 833); 4) urine pregnancy test positive(n = 147); 5) taking prednisone or progesterone(n = 238). 10710 participants were excluded for the above reasons.Data on potential confounders, such as gender, age, and ethnicity, derived from participant responses to questionnaires, total cholesterol(TC), high density lipoprotein (HDL), uric acid(UA), were derived from laboratory tests performed on fasting blood samples. Alcohol consumption was defined as 12 or more alcohol intakes per year. Smokers are defined as smoking 100 or more cigarettes in their lifetime. We divided smoking status into smoking, non-smoking, and former smoking according to two questions in the smoking questionnaire:“have you smoked at least 100 cigarettes in entire life?”and “do you now smoke cigarettes?”. Total physical activity(TPA) was based on the Global Physical Activity Questionnaire, which included minutes of recreational, occupational, transportation-related physical activity, and was divided into vigorous and moderate categories according to the intensity of each physical activity. Double the time of high-intensity physical activity and add it to the time of moderate-intensity physical activity ,16. The  obesity , which wTo demonstrate the independent effect of bedtime on diabetes, we also collected other sleep factors into the data analysis, including sleep duration, snort or stop breathing, and trouble sleeping. According to the American Academy of Sleep Medicine Statement on Recommended Sleep Amounts for Adults . Sleep dhttp://www.empowerstats.com) and statistical package R (http://www.R-project.org). The P value was taken as the cut point of 0.05 to indicate the statistical significance of the results.Given the complex survey design (including oversampling), survey non-response, and post-stratification, NHANES created sample weights. A sample weight was assigned to each survey respondent, and when a sample was weighted, its data were representative of the U.S. civilian noninstitutionalized resident population. The weights used for data analysis in this study were mobile examination center(MEC) exam weights and we have adjusted the MEC exam weights of the data for these two periods based on official guideline [Table 1. The sample sizes for diabetes(no) and diabetes(yes) were 11920 and 2901, respectively. In the diabetes(yes) group, the participants were more likely to be male(52.78%), older(54.36%), non-drinkers(47.60%), inactive participants(47.18%), obese(62.33%), and less likely to be smoker(13.93%), and the level of TC, HDL were much more lower. The sleep-related factors of the study subjects were shown in Table 2. In the diabetes(yes) group, the proportion of snort or stop breathing and trouble sleeping was 28.67%, 39.83%, respectively. 23.35% subjects with sleep duration less than 7 h. Fig 2 presented the unweighted distribution of different bedtime points for the study population. We can see from the figure that that most people’s bedtime were distributed at the four time points of 0:00, 23:00, 22:00 and 21:00.Grouping was based on the presence or absence of diabetes, and we described the baseline characteristics of the study subjects in Table 3 illustrated the results of a weighted multivariate logistic regression analysis between bedtime at night and adult diabetes incidence. In the model I, we adjusted for sleep-related factors: sleep duration, snort or stop breathing, trouble sleeping, into the model, a significantly negative association can be found between bedtime and diabetes. In Model II, Model III and Model IV, we gradually added social, lifestyle, and metabolic factors into the model adjustment, but the relationship was not statistically significant. The estimates and 95% confident intervals for each variable in Models I-IV can be found in  To explore the nonlinear relationship between bedtime and diabetes, we performed a smooth curve fitting. From the figure, we can see that the relationship between bedtime and diabetes was U-shaped from 19:00 to 02:00, and was lowest at 23:00. However, we can see from Table 4). From 19:00 to 23:00, a significantly negative association can be found between bedtime and diabetes. From 23:00 to 02:00, The relationship between the two was positive, nevertheless, the P values was not statistically significant(p = 0.3524). We also explored the effect of bedtime on diabetes in different genders. From 19:00–23:00, the relationship was negative across genders, and in males, the P-values were still statistically significant(p = 0.0414). From 23:00–02:00, the relationship was positive across genders. And there was no significant interaction of gender on the relationship between bedtime and diabetes at different time periods.This research performed on US adult population included 14821 subjects revealed that bedtime was significantly associated with the incidence of diabetes. Earlier bedtime (before 23:00) increased the risk of developing diabetes. And this effect was not significantly different between male and female. For bedtime between 23:00–2:00, although the data was not statistically significant, the risk of developing diabetes tended to increase as bedtime was delayed.The time of go to bed was a critical node in the sleep-wake cycle and light was the main synchronizer of the central circadian clock(suprachiasmatic nucleus). The suprachiasmatic nucleus then transmitted its signals through the neuroendocrine pathway to peripheral tissues to regulate the circadian clock in peripheral tissues, including: adipose tissue, muscle, pancreas, gut, and liver ,12,21,22According to our current knowledge, our study should be the first to clarify the relationship between bedtime and diabetes risk. And the conclusions of the study have characteristics that are representative of the population of the United States. We adjusted for diabetes-related risk factors as much as possible, and adjusted for other sleep factors: sleep duration, insomnia, and sleep apnea in order to highlight the effect of bedtime on diabetes. Of course, our study also has certain flaws. Firstly, due to the natural defects of the database, we could not distinguish type 1 diabetes and type 2 diabetes in the diabetic population of this study. In order to make the study conclusions more accurate, we excluded people younger than 18 years, to reduce the number of patients with type 1 diabetes. Similarly, some genetic factors of diabetes we cannot obtain from the database. Secondly, some covariates are based on participants’ self-reports and may deviate from the actual situation.Earlier bedtime (before 23:00) increased the risk of developing diabetes. And this effect was not significantly different between male and female. For bedtime between 23:00–2:00, the risk of developing diabetes tended to increase as bedtime was delayed.S1 Table(DOCX)Click here for additional data file.


Anoikis is a programmed cell death process that was proven to be associated with cancer. Uroepithelial carcinoma of the bladder (BLCA) is a malignant disease of the urinary tract and has a strong metastatic potential. To determine whether anoikis-associated genes can predict the prognosis of BLCA accurately, we evaluated the prognostic value of anoikis-associated genes in BLCA and constructed the best model to predict prognosis.CALR, FASN, CASP6, and RAD9A.The BLCA transcriptome data were downloaded from TCGA and GEO databases, and genes with differential expression were selected and then clustered using non-negative matrix factorization (NMF). The genes with the most correlation with anoikis were screened and identified using univariate Cox regression, lasso regression, and multivariate Cox regression. The GEO dataset was used for external validation. Nomograms were created based on risk characteristics in combination with clinical variants and the performance of the model was validated with receiver operating characteristic (ROC) curves. The immunotherapeutic significance of this risk score was assessed using the immune phenomenon score (IPS). IC50 values of predictive chemotherapeutic agents were calculated. Finally, we used RT-qPCR to determine the mRNA expression of four genes, We screened 406 tumor samples and 19 normal tissue samples from the TCGA database. Based on anoikis-associated genes, we classified patients into two subtypes (C1 and C2) using NMF method. Subsequently, nine core genes were screened by multiple methods after analysis, which were used to construct risk profiles. The design of nomograms based on risk profiles and clinical variables, ROC, and calibration curves confirmed that the model could well have the ability to predict the survival of BLCA patients at 1, 3, and 5 years. By predicting the IC50 values of chemotherapeutic drugs, it was learned that the high-risk group (HRG) was more susceptible to paclitaxel, gemcitabine, and cisplatin, and the low-risk group (LRG) was more susceptible to veriparib and afatinib.In summary, the risk score of anoikis-associated genes can be applied as a predictor to predict the prognosis of BLCA in clinical practice. ApproxiAnoikis is a form of programmed cell death that occurs when cells lose contact with the extracellular matrix . It is aIn this study, we investigated the role of anoikis-related genes in bladder cancer using data from the GEO and TCGA-BLCA cohorts. Specifically, we aimed to identify a set of genes associated with anoikis resistance that could be used to predict treatment response and prognosis. We also examined the potential signaling pathways involved in anoikis resistance and assessed the sensitivity of different chemotherapeutic drugs in relation to the risk score generated from our gene expression analysis. By addressing these questions, we hope to provide important insights into the prognostic value of anoikis-related genes in bladder cancer and highlight potential therapeutic targets for the disease.22.1Pathological samples related to BLCA and normal bladder tissue data were obtained from GSE31684 cohort and TCGA-BLCA dataset. After removing patients with no clinical information, 93 tumor samples were obtained in GSE31684. 406 cases of tumors and 19 cases of normal samples were selected from the TCGA-BLCA cohort. The transcriptome profiles of these samples were retrieved, and then 434 potential genes associated with anoikis were screened through the literature and Genecards database Table S12.2NMF was used instead of hierarchical clustering for tumor typing . The bio2.3The subtype abundance of 9 different immune cells and stromal cells that could represent the tumor immune microenvironment was obtained. Based on the microenvironment cell populations, infiltrating cells were identified by comparing scores between the two groups.2.4After using univariate Cox regression, genes associated with anoikis were identified. They were processed by lasso regression analysis and multivariate Cox regression analysis to prevent overfitting of these genes, and at the same time analyzing the prognostic characteristics of these genes, the risk score of each sample was determined using the formula equation:Here, coef was the regression coefficient in the multivariate Cox regression analysis as described previously. X indicated the expression of candidate genes. And the median risk score was used as the cut-off point, each sample was divided into high-risk group (HRG) and low-risk group (LRG), and the overall survival rate of the HRG and LRG was analyzed. The survival curves of the HRG and LRG of the related genes screened were analyzed separately.2.5Receiver operating characteristic (ROC) analysis was performed on risk scores and clinical variables at years 1, 3, and 5 to determine the best prognostic indicators. The R program “rms” was established and then “regplot” was used to complete the visualization. The calibration curve was used to evaluate the consistency of the model.2.6Gene set enrichment analysis (GAES) was used to label the gene function of the HRG and LRG, and the first 8 results were visualized, which were selected with the results’ statistically significant (P<0.05).2.7The distribution of clinical variables in HRG and LRG was visualized, and the proportion of patients in the subgroups of clinical variables was displayed.2.8Information on the corresponding somatic alterations was obtained from TCGA-BLCA. The 20 most common driver genes with frequent somatic mutations were presented. Based on the risk of TMB and the HRG and LRG of the samples, patients are divided into four major categories, and their survival rates are calculated separately to construct survival curves .2.9We used XCELL, TIMER, QUANTISEQ, MCP COUNTER, EPIC, CIBERSORT, and CIBERSORT-ABS to reveal the correlation between risk score and immune infiltrating cells .2.10The pathway of the core gene was analyzed using the KEGG database to assess the degree of activation of its signature pathway and metabolic pathways. Normalized GSVA was calculated for each gene set in each sample to determine the relative pathway activity, immune markers, and immune checkpoints for each gene set.2.11After obtaining immune checkpoint blockage-associated genes, their expression levels were explored according to the risk score. Immunophenoscore (IPS) uses the expression of genes associated with immune checkpoints to assess the immunogenicity of tumors in high- and low-risk samples.By constructing a cell expression family tree based on TCGA-BLCA data. For tumor drug susceptibility genomics, the R package “pRRophetic” was used to predict the sensitivity of tissues to different drugs.2.122 environment. The four different cell lines were subjected to real-time quantitative polymerase chain reaction (RT-qPCR), and the relative expression of CALR, CASP6, FASN, and RAD9A was calculated using the 2-ΔΔCt method with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels as the endogenous control. The primer sequences used for PCR were as follows: :CALR: F: CCTGCCGTCTACTTCAAGGAG R: GAACTTGCCGGAACTGAGAAC; CASP6: F: ATGGCGAAGGCAATCACATTT R: GTGCTGGTTTCCCCGACAT; FASN: F: AAGGACCTGTCTAGGTTTGATGC R: TGGCTTCATAGGTGACTTCCA; RAD9A: F: CATTGACTCTTACATGATCGCCA R: GCCAGGTGAAAGGGAAATGG.Four cell lines, UMUC3, RT112, T24, and SV-HUC1, were selected. Cells were cultured in MEM, RPMI-1640, 5A, and F-12K medium plus 10% fetal bovine serum, respectively, at 37°C in a 5% CO2.13The Kruskal-Wallis test was used to compare data from more than two groups, and Wilcoxon was used to compare data from two groups. Kaplan-Meier log-rank test was used to evaluate each survival curve. A chi-square test was performed to correlate risk score subgroups with somatic mutation frequency, and Spearman analysis was performed to calculate the correlation coefficient. The results of the CIBERSORT algorithm with p< 0.05 were used for further analysis. p< 0.05 was considered statistically significant. R software was used for all statistical analyses.33.1We obtained 406 tumor tissue samples with clinical information and 19 normal tissue samples based on TCGA-BLCA data. The Clinicopathological characteristics of BLCA patients from the TCGA and GEO databases were shown as 3.2The TCGA-BLCA transcriptome data were clustered by NMF method. The stability and clustering performance were based on cophenetic and RSS. When K=2, clustering was the optimal value, that was, the sample can be divided into two categories, C1 and C2  were identified in BLCA, all of which were considered prognostic indicators (CALR) - (0.3257*CASP6) + (0.0983*CCDC80) + (0.1480*CSPG4) + (0.3173*FASN) + (0.1698*HMGA1) - (0.0789*ITGA3) + (0.1792*RAC3) - (0.4413*RAD9A).We performed univariate Cox regression analysis and identified 25 genes with significant prognostic value , while FGFR3 had a higher somatic mutation rate in the LRG (19% vs 8%). These findings had the potential to benefit applications for anoikis in the treatment of BLCA.To investigate the correlation between risk scores and gene mutations, the top 20 gene waterfall plots that were most common among somatic mutations were shown  in patients with BLCA. Paclitaxel, gemcitabine, and cisplatin showed higher IC50 (p<0.001) in the HRG, and the responsiveness to veliparib and afatinib in the LRG was better than that in the HRG (P<0.001) (3.12CALR, FASN, CASP6, and RAD9A, respectively. The results showed that the gene expression of these three genes in cancer cells was significantly lower than that of normal cells, and the results were statistically significant. In summary, in cancer cells, anoikis-related genes were significantly inactivated, and these genes were somewhat associated with the progression of tumor cells (Four cell lines of T24, UMUC3, RT112, and SV-HUC1 were selected to verify the gene expression of 4BLCA is a highly malignant urinary tumor, and genomic therapies such as regulation of noncoding RNAs, DNA methylation, and gene loci mutations are key regulators that prevent the continued progression of BLCA , 15. TheCALR gene have found that CALR is associated with immune responses and apoptosis of cells (CALR is associated with cell carcinogenesis, drug resistance of tumor cells, and epithelial-mesenchymal transformation (Casp6 in cancerous tissues was low expression, indicating that it had a certain inhibitory potential in tumorigenesis and progression (Casp6 was inversely correlated with the IC50 value of 5fluorouracil (Casp6 is currently widely recognized as a key regulator of innate immuno-inflammatory activation and host defense (CCDC80 can enhance the sensitivity of tumor cells to chemotherapy drugs, and knocking it down in cancer cells can enhance the anti-cancer drug resistance effect of bladder cancer cells (FSAN contributed to the metabolic conversion and proliferation of tumor cells (Studies on the effects of the of cells . There iormation . The stugression . and therouracil , 20. Cas defense . At preser cells . In the or cells . In studor cells .Clinical practice has shown that patients with high TMB are more conducive to immune checkpoint inhibitor therapy, TMB classified according to anoikis-associated genes in this study have a longer survival time . The funIn short, anoikis plays a key role in the occurrence and development of tumors. Our study demonstrates the value of a group of anoikis-associated genes as biomarkers for BLCA prognosis. As a retrospective study conducted by bioinformatics analysis, the clinical application of this information is not perfect, and these biological mechanisms for predicting prognosis still need to be further confirmed.5In summary, we developed a model for predicting prognosis, immune microenvironment, and chemotherapy response in BLCA patients. The model was constructed based on nine anoikis-associated genes and clinical risk characteristics, and multidimensional validation of the model showed that the model has the potential to reliably predict clinical prognosis in BLCA species.The original contributions presented in the study are included in the article/CX, YD, GS and HM designed this work. YD, CX, YaL, BY, YuL, GS, TY, SM, HM contributed to data analysis. YD and CX wrote this manuscript. HM edited and revised the manuscript. All authors contributed to the article and approved the submitted version.


P < .001). The mean EQ-5D-5L score at follow-up showed a significant increase of 0.18 points compared with that at admission (P < .05). In the follow-up survey on PGIC, 307 participants (90.56%) were “minimally improved” or better. Integrative KM treatment can help improve pain, functional impairment, and long-term quality of life in patients with traumatic fractures.Previous studies have reported pain reduction after Korean medicine (KM) treatment in patients with fractures. However, these studies were limited by small sample sizes and short observation periods. To address these limitations, we aimed to analyze the outcomes of patients with traumatic fractures who received integrative KM treatment and investigate their long-term progress through follow-up observations. This study was a retrospective analysis and questionnaire survey conducted at a multi-center inpatient care setting in Korea. A total of 1150 patients who had traumatic fractures and received at least 5-day inpatient care at one of 5 KM hospitals. Finally, 339 patients completed the follow-up survey. The questionnaire survey was administered 3 months post discharge. The primary outcome was the difference in numeric rating scale (NRS) scores at admission and discharge for fracture-related pain. The secondary outcomes were EuroQol 5-Dimension 5-Level (EQ-5D-5L) score, Oswestry Disability Index, Neck Disability Index, Western Ontario and McMaster Universities Arthritis Index, Shoulder Pain and Disability Index, and Patient Global Impression of Change (PGIC) score. The follow-up questionnaire survey included questions on surgery and imaging before admission and after discharge and treatment within the past 3 months. The mean NRS score at follow-up showed a significant decrease of 4.41 points compared with that at admission ( Contributing to this trend are the aging population and an increased number of traffic accident victims. The number of patients undergoing treatment for fractures has increased consistently from 2,255,134  in 2016 to 2,503,502  in 2019. Moreover, fracture-related medical costs increased from 1,556,661,414 Won in 2016 to 2,093,066,351 Won in 2019.A fracture, which is a loss in the structural continuity of a bone or cartilage due to the excessive application of force, may be caused by an external force, such as trauma, or may be a result of a pathological condition that weakens bones, such as osteoporosis.In addition to the direct medical costs associated with fracture treatment, fractures can directly or indirectly cause other long-term problems. First, a fracture can lead to an additional fracture in another area or increase the risk of other chronic comorbidities. Moreover, fractures can lead to functional impairment and mobility loss, which are highly associated with depression, reduced quality of life (QoL), and loss of productivity.,6 In Korea, which has a dual healthcare system, non-surgical patients or patients undergoing postoperative bone union and rehabilitation may choose Korean medicine (KM) treatment as conservative treatment. KM is based on traditional treatment approaches; it combines Korea’s independent sa-sang theory with traditional Chinese medicine and primarily makes use of acupuncture, herbal medicine, and chuna manipulation.Fracture treatments, namely, conservative treatment and surgery, are determined based on the fracture pattern. Surgical intervention, including internal or external fixation and open reduction, is primarily applied in complex fractures such as open, comminuted, or multiple fractures. For non-complex fractures, conservative treatment may be applied, including closed reduction, fixation , analgesics , and nerve blocks. Another randomized controlled trial (RCT) revealed pain reduction and function recovery following acupuncture in 97 patients with intertrochanteric fractures. A systematic review on patients with humeral fractures showed significantly lower levels of pain in the acupuncture group than in the control group. However, several studies have included only small sample sizes, and case studies account for most of the studies to date. Meanwhile, follow-up observational studies on the long-term effects in patients with fractures are still lacking. Accordingly, the aim of this study was to analyze the outcomes of patients who received integrative KM treatment for traumatic fractures and examine the long-term progress of the therapeutic effects of integrative KM treatment.A retrospective observational study showed pain reduction after KM treatment in 17 patients with hip fractures.This multi-center follow-up observational study investigated inpatients with traumatic fractures. We retrospectively analyzed the medical records of patients with traumatic fractures who received at least 5-day inpatient care at 5 KM hospitals in Gangnam, Bucheon, Daejeon, Haeundae, and Bundang between March 2017 and August 2020. In addition, we conducted a follow-up questionnaire survey on patients who consented to take part in the survey. All 5 KM hospitals included in the study are accredited by the Ministry of Health and Welfare. Integrative Western medicine (WM) and KM treatments are administered in these hospitals based on modern medical diagnostic technologies.Patients aged 19–85 years who had a fracture caused by an injury; received at least a 5-day inpatient care at the Gangnam, Bucheon, Daejeon, Haeundae, or Bundang branch of Jaseng Hospital of Korean Medicine; who understood the objective and contents of the study; and who consented to participate in the survey were included.Patients with pathological or stress fractures; with open fractures caused by trauma; with pain caused by diseases other than fractures ; and who could not provide appropriate responses to the questionnaire were excluded. Patients with insufficient data who were determined to be unfit for participation by the researcher were also excluded.The patients or public were not involved in designing the study, recruitment, or study conduction. Additionally, there are no plans to announce the results of the study via any other platform.We collected the following medical information from the electronic medical records (EMRs): sex, age, and type of insurance; date of admission, date of discharge, length of hospital stay (in days), and diagnosis; previous medical history; date and cause of the onset of current symptoms; admission to a WM hospital prior to inpatient care; if so, treatment details; details of treatments received during inpatient care at the applicable KM hospital (type and frequency of treatment); numeric rating scale (NRS) score, EuroQol 5-Dimension 5-Level (EQ-5D-5L) score, Oswestry Disability Index (ODI), Neck Disability Index (NDI), Shoulder Pain and Disability Index (SPADI), and Western Ontario and McMaster Universities Arthritis Index (WOMAC) score for the fracture site from admission to discharge.Patients received treatments during their inpatient care according to the integrative KM treatment protocol. Treatment was determined by KM doctors based on patients’ individual conditions. Integrative KM treatments administered during inpatient care included acupuncture, electroacupuncture, pharmacopuncture, cupping, herbal steam therapy, chuna, and herbal medicine. All treatments were recorded in the EMRs, which were used for treatment investigation.A follow-up questionnaire survey was conducted after discharge to investigate the status of the fracture site and patients’ satisfaction with KM treatment. The survey was conducted via telephone calls or online using Google forms. The survey contents included recommendation of surgery before admission or after discharge, and if the patient had undergone surgery, the type of surgery; time frame of surgery; any radiological examination after discharge for reevaluation, and if so, radiological findings; treatment within the past 3 months, and if so, the type and frequency; Patient Global Impression of Change (PGIC) score and satisfaction with KM treatment; and current status of fracture site .Among the questions for assessing the fracture site’s status, the NRS and EQ-5D-5L were implemented in all patients regardless of the fracture site, while all other questions were tailored according to each patient’s fracture site.NRS is a numeric scale, ranging from 0 to 10, used to rate the severity of pain, with 0 representing “no pain” and 10 representing “the worst possible pain.” The NRS scores of the fracture sites measured every 2 weeks from admission to discharge were obtained from the EMRs and were also measured during the follow-up survey. The effectiveness of integrative KM treatment was assessed based on comparison of the NRS scores between admission and discharge.For the EQ-5D-5L, ODI, NDI, WOMAC, and SPADI, the scores measured every 2 weeks from admission to discharge were obtained from the EMRs, and the scores were additionally measured during the follow-up survey. Where possible, the Korean version, with proven validity and reliability, of each index was used.,13The EQ-5D-5L, consisting of 5 items , is used to assess patients’ QoL. Each item is rated on a 5-point scale. The Korean version was used.,15The ODI, consisting of 10 items, is designed to assess the functional status of patients with low back pain. Each item consists of 6 levels, ranging from 0 to 5 points, with higher scores indicating more severe disabilities. The Korean version was used for patients with lumbar, thoracic, or sacral spine fractures.,17The NDI, consisting of 10 items, is designed to assess neck disability. Each item consists of 6 levels, ranging from 0 to 5 points, with higher scores indicating more severe disabilities. The Korean version was used for patients with cervical spine fractures.,19The WOMAC, consisting of 3 subscales , is designed to assess knee joint function. Each item is rated on a 5-point scale, with higher scores indicating a poorer knee joint status. The Korean version was used for patients with knee fractures.,21The SPADI, consisting of 2 dimensions , is designed to assess shoulder pain and disability. Each question is rated on a scale of 0 to 10 points, with higher scores indicating more severe shoulder disabilities. The Korean version of the SPADI questionnaire was used for patients with shoulder fractures.,23The Q-DASH is a shortened version of the DASH and is designed to assess the function of patients with upper extremity disorders. It consists of 11 basic questions and 2 optional scales . Each question is rated on a scale of 0 to 5 points, with higher scores indicating more severe upper extremity disabilities. We used the Korean version. Patients with fractures of the clavicle, arm, elbow, wrist, or hand were evaluated based on the basic questions of the Q-DASH during the follow-up survey.,25The FFI is designed to assess the impact of foot pain on the activities of daily living (ADL). It consists of 3 subscales  with 9 items each. The Korean version was used and had only 2 validated subscales (pain and disability). Each item was rated on a scale of 0 to 9 points, and the total score for each subscale was converted to a scale of 0 to 100 points, with higher scores indicating poorer foot functions. In this study, only the 9 items in the pain subscale of FFI were used to evaluate patients with fractures of the foot or ankle during the follow-up survey.,27HOOS is designed to assess the extent to which hip problems impact ADL. It includes 10 items on pain, 5 items on symptoms, 17 items on ADL, 4 items on sport and recreation, and 4 items on QoL. Each item consists of 5 levels, from 0 to 4 points, and the total score for each subscale was converted to a scale of 0 to 100 points, with lower scores indicating better status. The Korean version was used to evaluate patients with hip fractures, including fractures of the pelvis, ilium, ischium, or pubic bone, during the follow-up survey.: deep breathing, coughing, turning over the body, and sleeping. Each item consisted of 4 levels.The following 4 items were included in the survey based on typical symptoms that appear in cases involving rib or sternal fractures, as reported in a previous studyDuring the follow-up survey, PGIC was used to investigate the patients’ subjective opinions regarding fracture-related pain and functional improvement. It is used for the subjective assessment of the impression of change based on 7 levels.This study was approved by the Institutional Review Board of Jaseng Hospital of Korean Medicine  and was registered at ClinicalTrials.gov (NCT04855058). The EMRs of patients who had given prior consent to use their medical information for research purposes were analyzed. The follow-up questionnaire survey was conducted for discharged patients who consented to survey participation after a full understanding of the use of their personal information. If any patient refused to provide consent for the study, the use of their personal information was discontinued, and such information was destroyed immediately.For all statistical analyses, two-sided tests were performed, with the significance level set at 5%. Continuous variables are presented as means and standard deviations, while categorical variables are presented as frequencies and percentage. The fractures were repeatedly measured from at least one site on each patient, while a linear mixed model that included subject random intercept was used to test the outcomes with respect to a decrease from the baseline. The outcomes  at admission, discharge, and the follow-up survey were expressed as least square estimates.–32 Discharge was considered censoring, while patients with any missing values after the baseline were considered to show no MCID until discharge.Moreover, a survival analysis was performed to estimate the mean time until achieving a minimal clinically important difference (MCID) in the NRS. The MCID was considered a 30% decrease relative to the baseline NRS score.The length of hospital stay, intervention, and herbal medicine prescription from the EMRs were assessed using a frequency analysis. In the questionnaire survey, all survey items, except the assessment variables, were analyzed using a frequency analysis.This study included 1150 patients who received at least 5-day inpatient care for traumatic fractures at one of the 5 KM hospitals included in the study. Patients who did not meet the inclusion and exclusion criteria, those who refused the use of personal information or registered on the do-not-call list, duplicate patients with multiple hospitalizations, and those with no baseline NRS score were excluded. Subsequently, of the 665 included patients, 339 participated in the follow-up survey , and the mean age was 56.9 ± 16.4 years. Regarding the type of insurance reported on the questionnaire, automobile insurance was the most common response , followed by national health insurance . The mean duration from onset to hospitalization was 27.8 ± 163.1 days. Overall, 537 patients (80.8%) had visited a WM hospital before admission to a KM hospital, among which 316 patients (47.5%) were diagnosed with fractures at a WM hospital. There were 39 patients (5.9%) who had undergone surgery, with pin fixation  being the most common type of surgery. Moreover, 33 (5.0%) and 49 patients (7.4%) were treated with casts and splints, respectively  and 4.41 points , at discharge and follow-up, respectively, from that at admission. A subgroup analysis was performed for fracture sites with sufficient sample sizes: the ribs/sternum, lumbar spine, thoracic spine, ankle/foot, wrist/hand, and knee. The results showed a significant post-treatment reduction in NRS scores at all 6 fracture sites (P < .001).The mean NRS score for all fracture sites significantly reduced by 2.05 points  and 39.26 points  at discharge and follow-up, respectively, from that at admission. The mean WOMAC significantly decreased by 8.69 points  and 42.23 points  at discharge and follow-up, respectively, from that at admission. The mean EQ-5D-5L score significantly increased by 0.12 points  and 0.18 points  at discharge and follow-up, respectively, from that at admission . For fractures of the rib (n = 115) and sternum (n = 26) investigated during the follow-up survey, assessment of symptoms for 4 items  showed responses in the order of “not interfered at all” and “slightly interfered” . Regarding the scores obtained during the follow-up survey, the Q-DASH (n = 42) score was 10.12 ± 15.48 points, and the FFI (n = 23) was 12.65 ± 14.65 points. The scores for each subscale of the HOOS (n = 6) were 78.3 ± 22.1 for pain, 79.2 ± 19.6 for symptoms, 82.4 ± 20.5 for ADL, 77.1 ± 22.9 for sport and recreation, and 70.8 ± 23.6 for QoL.The NRS score, NDI, and SPADI for fractures of the sacrum, cervical spine, shoulder, clavicle, arm, and pelvis could not be statistically analyzed owing to the small number of cases, but the scores showed an overall decreasing trend . The median time for the knee was 14 days (95% CI: 9–NA), which was the fastest, followed in order by 19 days (95% CI: 14–21) for the lumbar spine, 21 days (95% CI: 19–23) for the rib/sternum, 21 days (95% CI: 19–NA) for the hand/wrist, 27 days (95% CI: 21–40) for the thoracic spine, and 27 days (95% CI: 21–NA) for the ankle/foot , and the patients received integrative KM treatment, including acupuncture, pharmacopuncture, and herbal medicine, during their inpatient care. The most frequently prescribed interventions were acupuncture, cupping, electroacupuncture, and pharmacopuncture . The most frequently prescribed herbal medicines were Ansinjitong-tang, Jeopgol capsules, and Gwanjeol-go .The mean length of hospital stay was 22.5 ± 13.9 days , and of these, surgery was performed before admission in 30 cases (8.11%). The most commonly performed surgery was pin fixation , followed by cement augmentation . The number of cases recommended for surgery after discharge was 11/370 cases (2.97%). The most commonly performed surgery was cement augmentation , followed by pin fixation .Additional radiological examination after discharge for progress assessment was performed in 163 cases (44.05%), of which findings showed complete recovery in 96 cases (25.95%), recovery with good progress in 56 cases (15.14%), and the need for additional treatment or surgery in 10 cases (2.70%). The number of patients who received treatment within 3 months from the survey was 53/339 (15.63%), and the mean number of treatments received in 3 months was 18.88 ± 10.65. The most frequently prescribed interventions were acupuncture , physical therapy , and cupping .For PGIC after KM inpatient care, the responses of 307 patients were “minimally improved” or better, accounting for 90.56% of all responses Table .In this study, assessment of various indicators of pain, functional impairment, and QoL showed improvement in patients with traumatic fractures who received integrative KM treatment. Moreover, analysis of the long-term therapeutic effect through a follow-up survey showed that the patients exhibited relatively good post-discharge progress. Q-DASH scores of 10.12 in this study can be viewed as indicating a relatively recovered state.In the post-treatment analysis, the results showed decreased NRS, ODI, WOMAC, and EQ-5D-5L scores at discharge and during the follow-up survey, compared with the scores at admission. The Q-DASH scores, HOOSs, and FFI, which were investigated for outcomes in each site during the follow-up survey, could not be compared and analyzed, as they were not measured at discharge. However, considering that a study on patients with humeral shaft fractures viewed Q-DASH scores < 15 as recovery,In this study, MCID was set as a 30% decrease in NRS scores, and the time required to achieve MCID or better was measured. Considering that the median survival time for all fractures was 21 days (95% CI: 20–21) and the mean length of hospital stay was 22.5 ± 13.9 days, it was determined that half of the patients showed improvement with a ≥ 30% decrease in NRS scores before discharge. In the analysis of inpatient care, the length of hospital stay was the longest with fractures of the thoracic spine, followed by the pelvis, lumbar spine, and sacrum, while fractures of the trunk tended to require longer hospital stays than fractures of the limbs.Per the follow-up survey, 46 cases (12.43%) were recommended surgery before admission, and among these, 30 cases underwent surgery (8.11%). Meanwhile, 13 cases (3.51%) were recommended surgery after discharge, and among these, 11 actually underwent surgery (2.97%). Among 16 cases (4.32%) in which the patients were recommended surgery before admission but did not undergo surgery, patients did not receive a recommendation for surgery after discharge in 13 cases (3.51%). Among the 3 cases which were recommended surgery after discharge, only 2 (0.54%) underwent surgery. The number of patients who had received treatment within 3 months of the follow-up survey was 53 (15.63%), while 286 patients (84.37%) did not receive treatment during that period. Regarding PGIC items, 307 (90.56%) patients responded “minimally improved” or better after integrative KM treatment. Based on such findings, it was determined that the conditions of most of the patients were maintained to the point of not requiring any additional treatment when the follow-up survey was conducted.,34–36The fracture healing process typically comprises 3 phases: the inflammatory, reparative, and remodeling phases. The inflammatory phase lasts for ~7 days after the fracture, during which time, hematomas and inflammatory exudates are formed from the ruptured blood vessels. Inflammatory cells and fibroblasts are mobilized, and hematoma at the fracture site is gradually replaced by granulation tissues, while osteoclasts replace bone tissue at the margins of bone fragments. The reparative phase is also referred to as the callus phase. During the soft callus phase, ~3 weeks after the fracture, pain and swelling subside and soft calluses form, while infiltration of blood flow and capillaries into the calluses and the number of cells increase. Subsequently, during the hard callus phase, the soft calluses are converted into hard calcified tissue through endochondral and intramembranous ossification. The remodeling phase starts after hard union of the fracture and continues for several months to years until the bone returns to its original form.,38 Measurement of radical scavenging activity of herbal extracts showed a dose-dependent increase in antioxidant activity and a significant decrease in the production of the inflammatory mediator interleukin-1β, indicating that herbal extracts can help relieve inflammatory responses accompanying fractures.,40 Osteogenesis during the reparative phase can be facilitated by bone morphogenetic protein type 2 that mediates the differentiation of osteoblasts and activates transforming growth factor-β1 and Sox9 that regulates the transcription of type 2 collagen. Moreover, bone union during the reparative phase and maintenance of stability during the remodeling phase can be increased through the collagen type II alpha 1 chain that regulates endochondral ossification,41 and expression of osteocalcin that contributes to bone remodeling and the change to hard calluses.,42The KM treatment approach aids in reducing pain and promoting recovery during the healing process. Herbal medicine is considered to be helpful in fracture healing processes. Previous experimental studies on herbal medicine showed that callus formation occurred faster in the herbal medicine group than in the control group. A study on only patients with hip fractures showed that taking herbal medicine reduced the risk of mortality, rehospitalization, and re-operation. In an RCT including 170 patients with rib fractures, the treatment time and cost until the visual analog scale (VAS) score was reduced by half were lower in the herbal medicine group than in the nonsteroidal anti-inflammatory drugs group. In this study, the most frequently prescribed herbal medicines were Ansinjitong-tang , Jeopgol capsules , and Gwanjeol-go . Ansinjitong-tang is effective in treating distal radius fractures, while Jeopgol capsules and Gwanjeol-go are effective in treating lumbar burst fractures.According to a study that used the Taiwanese National Health Insurance Research Database, the fracture patient group that received herbal medicine treatment had lower inpatient care costs within 6 months from the accidents than the control group. An RCT on the efficacy of acupuncture for intertrochanteric fracture revealed that the group that received a combination of acupuncture and aspirin showed significantly lower VAS scores at postoperative days 5 and 7 and significantly higher Harris scores and modified Barthel indexes 2 months postoperatively, than the control group that received only aspirin. A systematic review of patients with humeral fractures indicated that the acupuncture group showed significantly lower pain indicator scores than the control groups using conventional drugs such as analgesics, physical therapy, or placebos. Such findings suggested that acupuncture alone or in combination with conventional treatment can help in pain alleviation and recovery of function and daily living ability. The mechanism by which acupuncture helps with recovery from fractures has been reported to involve inhibition of free oxygen radicals and regulation of antioxidant enzymes and reduction in the levels of inflammatory factors, such as C-reactive protein and tumor necrosis factor-α.Several studies have been published on the effects of acupuncture for various fracture sites. A study that analyzed Taiwanese National Health Insurance Research Database for the effects of acupuncture on patients with hip fractures showed that the patient group that received 6 sessions of acupuncture treatment after fractures showed significantly lower mortality, rehospitalization, and re-operation rates than its counterparts.,49 In a retrospective review of patients with thoracic or lumbar compression fractures, the group that received complex pharmacopuncture therapy showed a significant decrease in VAS scores and a significant increase in ODI, compared with the control group that received only KM treatment.Most studies on pharmacopuncture for fractures used pharmacopuncture combined with other treatment interventions. Consequently, it is difficult to analyze the sole effects of pharmacopuncture. In an animal study using an intervention combining pharmacopuncture and herbal medicine, the group that received both pharmacopuncture and herbal medicine showed a significant increase in serum alkaline phosphatase levels compared with the control group that did not receive any intervention. In addition, increased osteogenesis and higher transforming growth factor-β1 expression in the fracture site were found in histological observations.The present study population included 665 patients, but only 50.98% (n = 339) took part in the follow-up survey. There is a limitation in that the effects by gender or age could not be compared due to the different fracture sites and low follow-up rates. The median time from discharge to the follow-up survey was 784 days, while the maximum was 1582 days. Therefore, the possibility of recall bias for surgery and treatment after discharge cannot be dismissed. Moreover, because the records regarding inpatient care were retrospectively reviewed, it was difficult to obtain data regarding the type of fracture or displacement. Among the outcome tests, surveys using questionnaires such as Q-DASH, FFI, and HOOS were not performed during hospitalization, and as a result, comparative analysis with pretreatment data was impossible. Due to this, there are limitations to reporting the effect primarily on the NRS and not fracture-specific questionnaires or area-function-specific questionnaires. In addition, it was analyzed by setting the MCID of NRS to 30%, but this is a setting based on chronic pain, and it was difficult to set an MCID that was suitable for fracture. Other limitations of this study included the fact that the study was not conducted in a controlled environment and that there was no control group as a comparator. The integrative KM treatment applied in this study included several different interventions that were applied together, and as a result, it was difficult to distinguish the therapeutic effects of each intervention. It was difficult to clearly distinguish the natural process of fracture healing from the additional effects of treatment. Furthermore, our inability to completely exclude the effects of other treatments received during the follow-up was a limitation of this study, even though treatment history within 3 months of filling the questionnaire was investigated. Moreover, studies on KM treatment for fractures are also lacking. Through this study, the treatment modes for fractures administered in KM hospitals, such as fracture sites and duration of treatment, were identified, and the sustained effects of KM treatment were identified through a follow-up survey. Furthermore, various assessment indicators, including the EQ-5D-5L scores, questionnaires for different fracture sites, and questionnaires on satisfaction, were used in addition to the NRS for multi-dimensional assessment of the effectiveness of integrative KM treatment. Therefore, the study findings could be used as reference data for further studies. To establish evidence for the effectiveness and safety of KM treatment for fractures, registry studies or RCTs using patients undergoing other surgical techniques or conservative therapy  as control groups are needed.Under Korea’s dual healthcare system, patients with fractures often choose KM treatment as they await natural bone union, if they do not need surgery or to improve pain and function in the fracture site postoperatively. Among all patients with fractures, the proportion of those who receive treatment at KM institutions has gradually increased, from 1.02% in 2016 to 1.38% in 2020; however, the percentage is still low.In conclusion, integrative KM treatment as a conservative treatment might improve the pain, function, and QoL in patients with traumatic fractures, and its sustained long-term effects were identified through a follow-up survey. Although further research with a high level of evidence is warranted, integrative KM treatment can be considered as a conservative treatment modality for the management of traumatic fractures.Conceptualization: Yoon Jae Lee, Jinho Lee.Formal analysis: Eun-San Kim.Methodology: Eun-San Kim.Supervision: Jinho Lee, In-Hyuk Ha.Writing – original draft: Min Kyung Kim, Kyoung Sun Park.Writing – review & editing: Gyu Cheol Choi, Jae Eun Yu, Hee Won Lee, Yong Su Kwon, Hyo Seung Huh, Suna Kim.


Recently, applied technology in the form of the combination of a probiotics and a digital poultry system, with the convergence of Information and Communications Technology and farm animals, has enabled a new strategy to overcome the livestock production crisis caused by climate change, while maintaining sustainable poultry farming in terms of care, feeding, and environmental management systems for poultry. The aim of this study was to investigate the biological mechanisms of animal behavioral welfare and production improvement using the combination of a probiotics and a digital poultry system in broiler chickens. A total of 400 one-day-old male broilers (ROSS 308) were randomly divided into four treatment groups, with five replicates each (20 birds/replicate pen) in a completely randomized design: control group with a conventional poultry system without probiotics (CON), conventional poultry system with 500 ppm of probiotics (CON500), digital poultry control system without probiotics (DPCS), and digital poultry system with 500 ppm of probiotics (DPS500). All experimental animals were reared for 35 days under the same standard environmental conditions. The experimental results indicated that the animal behavioral welfare, which includes drinking, eating, locomotion, grooming, and resting, in addition to foot pads, knee burns, plumage, and gait scores, as well as the growth performance of the broiler chickens, were improved by maintaining immune function and cecal microbiota balance via interaction between the combination of a probiotics and a digital poultry system. In conclusion, it was found that the combined system showed improved broiler growth performance and animal behavioral welfare. Thus, further studies of molecular biological mechanisms by the use of such a combined system to improve the nutritional composition and quality of chicken meats are recommended. B. subtillus, S. galilaeus, and Sphingobacteriaceae has been reported to improve egg production, egg quality, and broiler production, while reducing odor [The changes to animal feeding management and environmental conditions arising from global warming can increase animal stress levels greatly and affect animal behavioral welfare, which can lead to the loss of farm animals and food crises . Probioting odor ,5.Probiotics and digital livestock systems have been introduced as sustainable livestock strategies to help combat climate change ,6. Such In some previous studies, digital livestock systems were reported for improving egg production, egg quality, and animal behavioral welfare through increased nutrient digestibility, as well as balanced blood parameters, immune function, cecal microbiota, and short-chain fatty acids in laying hens and swine ,9. The dThe health of broiler chickens depends primarily on animal management and environmental condition control. As factory-intensive conventional livestock systems have fallen behind in terms of automation and insufficient labor, the overall control of poultry houses has been unsatisfactory. Moreover, a poor environment has negative effects on the animal behavioral welfare, which results in increased animal stress and impaired welfare, leading to reduced poultry production ,16,17.Until recently, little has been reported in the literature about the biological mechanism of the combination of a probiotics and a digital poultry system for improving animal behavioral welfare such as drinking, eating, locomotion, grooming, resting, and also foot pads, hock burns, plumage, and gait scores, in addition to growth performance such as body weight, feed intake, and the feed conversion ratio of broiler chickens ,9,10,18.However, based on the previously studied biological mechanism for improving the productivity and behavioral welfare of farm animals, applying the combination of a probiotics and a digital poultry system may have a positive effect on the growth performance and behavioral welfare of broiler chickens. Therefore, the study hypothesized that the application of this combined system will improve behavioral welfare and growth performance in broiler chickens. The aim of this study was to identify the biological mechanisms for improving immune functions, cecal microbiota balance, animal behavioral welfare, and growth performance in broiler chickens by using the combination of a probiotics and a digital poultry system.This study was conducted according to the guidelines for scientific and ethical procedures provided in the European Experimental Animal Handling License (SCT-w94058) and European Union Council Directive 2008/120/EC. The Scientific Research Ethics Committee of the Ministry of SMEs and Startups (MSS) of the Republic of Korea approved all the animal tests conducted in this study (Ethics Reference No: E-2035).2). The probiotic was prepared by combining 2% of Bacillus subtillus, Streptomyces galilaeus, and Sphingobacteriaceae (isolated from earthworm cast) with 98% earthworm cast. These products were provided by HooinEcobio Co., Ltd. . Each strain contained at least 3.5 × 108 colony-forming units per gram (CFU/g product) of the product [The animal experiments were performed in poultry research farms, the HooinEcobio Institute, Hongseong, Republic of Korea, 2021. Four-hundred healthy one-day-old male broiler chicks  were purchased from Hanyang hatchery . The animals were allocated between four treatment groups with five replicates each in a completely randomized design for 35 days as follows: control group with a conventional poultry system without probiotics (CON), conventional poultry system with 500 ppm of probiotics (CON500), digital poultry control system without probiotics (DPCS), and digital poultry system with 500 ppm of probiotics (DPS500). The animals were placed in five replicate pens with 20 broiler chickens per pen , Gumboro disease  at 21 days of age), and infectious bronchitis disease (IBD at 14 and 22 days of age) according to manufacturer instructions .The optimal environmental conditions, including temperature, humidity, and lighting, were maintained according to the Ross strain guidelines, as well as the Korean standard brooding practices and standard operating procedures ,21. The The study used a digital poultry system , which c2 gas (concentration of 40% or less), and their immune organs and cecum were collected. The spleen, thymus, and bursa of Fabricius index (as g per 100 g live body weight) are indicated. The cecum was collected and frozen in liquid nitrogen for storage at −80 °C to perform the bacterial counts.Live body weights and feed intake were recorded weekly for 35 days of the experimental period. The growth performance was then calculated based on the body weight, feed intake, and feed conversion ratio (weight gain/feed intake) at the beginning and the end of the experiment (35 days). At the end of the feeding experiments, the chickens were euthanized using COTo evaluate the immune biomarkers of the animals, about 8 mL of blood was collected from the jugular vein of each bird at the end of the experiment and placed in a clot activator vacuum tube . The serum corticosterone (a stress hormone) level was quantified using a chicken corticosterone ELISA Kit  according to the manufacturer’s manual. The serum immunoglobulin G (IgG) level was determined using a chicken cortisol ELISA Kit . Three blood smears per animal were obtained immediately after the blood collection, and the slides were stained using May-Grunwald-Giemsa after air drying. The heterophils (H) and lymphocytes (L) were differentiated using light microscopy. After 100 leukocytes per slide and 200 cells per animal were counted, the heterophil/lymphocyte ratio (H/L) was calculated by dividing the number of heterophils by the number of lymphocytes .1 to 10−9. Then, 100 µL of each diluted sample was plated on Difco™ Lactobacilli MRS agar for Lactobacillus, nutrient agar for total aerobic bacteria, and Violet red bile agar with MUG  for E. coli and coliforms, respectively. The Lactobacillus was incubated for 48 h at 37 °C under anaerobic conditions. Coliforms, E. coli, and the total aerobic bacteria were incubated for 24 h at 37 °C under aerobic conditions. The data are presented as log 10 colony-forming units (CFU) per gram of cecal content.To measure the cecal bacteria counts, the cecum collected at the end of the experiment was immediately placed in an anaerobic jar  equipped with an aerogen sachet . The cecum (1.0 g) was homogenized with 10 mL of sterilized 0.1 M phosphate-buffered saline at pH 7.0. The homogenate was serially diluted from an initial 10® Consortium [Observations of the behaviors and welfare of broiler chickens were based on the scientific concepts of animal behavior and welfare as per the Welfare Qualitynsortium . Animal nsortium  for drinnsortium . The plunsortium . The gainsortium . The foop < 0.05).Statistical analysis was performed by using a PROC GLM procedure in SAS Version 9.2.  under a The model used is:p < 0.013 to p < 0.025) or a digital poultry system (p < 0.012 to p < 0.015). There was an interaction effect between the probiotics and the digital poultry system (p < 0.015 to p < 0.020) (Significant differences in the growth performance were observed during the entire experiment because of the main effects of a probiotics (< 0.020) . The bodp < 0.011 to p < 0.027) and the digital poultry system (p < 0.007 to p < 0.020) or the interaction effect between the probiotics and the digital poultry system (p < 0.020 to p < 0.031); however, there were no differences between the DPCS and CON500 groups  or the digital poultry system ((p < 0.012 to p < 0.020). The interaction between the probiotics and the digital poultry system also had a significant effect on the immune function (p < 0.011 to p < 0.031) (p < 0.05). In contrast, there was no difference between the DPCS and CON500 groups. The serum IgG levels were significantly higher in the DPS500 group than the other groups (p < 0.05), and there was no difference between the DPCS and CON500 groups. The serum corticosterone levels and heterophil to lymphocyte ratios (H/L) were significantly highest in the CON and lowest in the DPS500 groups (p < 0.05), and no differences were noted between the DPS500 and DPCS groups.The immune biomarkers of the broiler chickens were significantly the highest in the DLS500 group raised with the combination of the probiotics and the digital poultry system because of the main effects of the probiotics (< 0.031) . Furtherp < 0.019 to p < 0.031) or the digital poultry system (p < 0.010 to p < 0.025). There was an interaction effect between the probiotics and digital poultry system (p < 0.015 to p < 0.031) , while the cecal microbiota showed no differences between the CON500 and DPCS groups.The cecal microbiota had significant differences observed at the end of the experiment (35 day) because of the main effects of the probiotics (< 0.031) . Cecal Lp < 0.015 to p < 0.025) or the digital poultry system (p < 0.010 to p < 0.030). In addition to, there was an interaction effect between the probiotics and digital poultry system (p < 0.015 to p < 0.030) , drinking, resting (lying down), and grooming, as well as the body condition scores of the foot pads, hock burns, plumage, and gait of the broiler chickens were significantly higher in the order of the DPS500, DPCS, CON500, and CON groups because of the main effects of the probiotics (< 0.030) . HoweverRenewed interest in the application effects of a digital poultry system along with probiotics has gained global attention in recent years owing to the convergence of livestock and ICT tools as sustainable livestock farming strategies in response to climate change ,2,9,11. The study results indicate that the combined system can suppress animal stressors by stimulating immune functions in broiler chickens. In addition, one of the most-important roles of probiotics is to stimulate immunity against invading pathogenic bacteria; they are known to improve the appropriate microbial environment of the digestive tract and host animal immunity ,29,30. AThese results found with respect to broiler chickens may be attributable to the use of smartphones through sensing platforms, automatic animal feeding management, and environmental management via central control through cloud computing systems ,7,10. ItLactobacilli, E. coli, and total aerobic bacterial balance, but the DPCS and CON500 group showed more balanced results than the CON group. The digital poultry system maintains gut microbiota balance compared to the CON group . In stressful environments such as poor health of the animals, heat and cold, digestive disorders, reduced nutrient availability, and reduced immune function [Our results suggest that the intestinal microbiota balance was well maintained in broilers raised under a combination of a probiotics and a digital poultry system. The gut microbiota are directly related to the growth performance and health of animals. In general, the number and structural composition of gut microbiota play important roles in the absorption of nutrients and health in the host animals . There wON group . The gutON group  and actiON group . Even thON group . Lactobanditions ,3. In thnditions ,3,28,30.nditions ,9,10,11.function ,15, stabfunction ,29,32. Ifunction ,33.The study results showed that the combination of a probiotics and a digital poultry system can improve animal behavioral parameters, such as drinking, eating, locomotion (moving), grooming, and resting (lying down) as well as animal welfare indexes, such as the foot pads, knee burns, plumage, and gait, compared to the conventional poultry system . DespiteAn ICT-converged digital poultry system using AI, big data, cloud computing, IOT, ML, and multimodal sensor technologies can guarantee greater amounts of free animal rights by reducing stress, which is related to animal behavioral welfare ,9,10,11.In conclusion, the study found that the combination of a probiotics and a digital poultry system could improve animal behavioral welfare and the growth performance of broiler chickens, as reported in the text. The results showed that the combined system was able to maintain an animal homeostasis balance such as immune biomarkers and cecal microbiota. In addition, growth performance was improved by enhancing the behavioral welfare scores and body condition scores in the broiler chickens. Although the study did not find a significant effect between the conventional poultry system with the probiotics and the control group of the digital poultry system without probiotics, there was a better balance of animal homeostasis in broiler chickens raised using the probiotics in the digital poultry system. This can be seen as a biological mechanism that appeared through the balanced interaction between the functional characteristics of the probiotics and the enabling of livestock management and environmental control monitoring through remote control and sensing technology by the digital poultry system, which is a convergence technology of ICT and livestock farming.