The Xinjiang autonomous region, located in west China, has a unique ethnic structure and a well-developed livestock industry. People in this region have a high risk of exposure to antibiotics, but the exposure level to antibiotics in relation to dietary determinants is unknown. In this study, 18 antibiotics, including four human antibiotics (HAs), four veterinary antibiotics (VAs), and 10 preferred veterinary antibiotics (PVAs) were detected in the urine of approximately half of the 873 adults in Xinjiang, including Han Chinese (24.6%), Hui (25.1%), Uighur (24.6%), and Kazakh (25.7%). Logistic regression was used to analyze the association between antibiotic exposure levels and adult diet and water intake. The detection percentage of antibiotics in the urine of adults in Xinjiang ranged from 0.1% to 30.1%, with a total detection percentage of all antibiotics of 49.8%. HAs, VAs and PVAs were detected in 12.3%, 10.3%, and 40.5%, respectively. Fluoroquinolones were the antibiotics with the highest detection percentage (30.1%) and tetracyclines were the antibiotics with the highest detected concentration (17 ng/mL). Adults who regularly ate pork, consumed fruit daily, and did not prefer a plant-based diet were associated with thiamphenicol, norfloxacin, and fluoroquinolones, respectively. These results indicated that adults in the Xinjiang autonomous region were extensively exposed to multiple antibiotics, and some types of food were potential sources of exposure. Special attention should be paid to the health effects of antibiotic exposure in humans in the future. Spatial modeling estimates that cumulative global consumption of antibiotics increased by 46% from 2000 to 2018 ,4. In adThe potential adverse effects on human health of continuous exposure to low doses of antibiotics in the environment have begun to attract the attention of scholars in the research community. A number of studies has investigated antibiotic exposure in humans by analyzing antibiotics in biological samples, and extensive antibiotic exposure was reported ,7,8. XinExposure to antibiotics can occur through food consumption and may be associated with sociodemographic factors. It is unclear which factors are most associated with antibiotic concentrations. We sought to investigate the antibiotic exposure profile in adults in Xinjiang and to explore the potential determinants of urinary antibiotics in adults using biomonitoring data of urinary antibiotics.Since 2018, our group has conducted the National Key Research and Development Program “The Xinjiang Multiethnic Cohort (XMC) study” in Xinjiang . A sub-sThe baseline questionnaire of the Northwest Natural Population Cohort Study was used to investigate the demographic characteristics , dietary status , and lifestyle . All questionnaires were completed by formally trained enumerators.Medical information included body mass index (BMI), hospitalization information, and antibiotic use within one month. Body measurements were obtained according to standard anthropometric measurements, with height and weight readings accurate to 0.1 cm and 0.1 kg, respectively; waist circumference was taken at the midpoint from the upper edge of the hip bone to the lower edge of the rib cage on both sides, and measured during calm exhalation, with a reading accurate to 0.1 cm; hip circumference was measured as the circumference of the widest part of the hip on the horizontal plane, with a reading accurate to 0.1 cm. Body fat percentage and muscle mass were measured by bioelectrical impedance . Blood pressure was measured at rest using an electronic sphygmomanometer (model: Omron HEM-7133) at the brachial artery of the right upper arm of the study subjects.Eighteen antibiotics from five major classes commonly detected in previous studies due to possible human exposure were measured ,11. ThesMorning urine 12 mL) was collected from the study participants and the samples were stored on site in a −40 °C refrigerator in the dark and frozen after collection. Prior to analysis, 1 mL of the urine sample was taken and sent to the laboratory for analysis. The concentrations of the 18 antibiotics in urine were determined using ultra-performance liquid chromatography coupled with high-resolution quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS) [ mL was cXinjiang is a multi-ethnic region, with Han Chinese, Uyghurs, and Kazakhs accounting for more than 90% of the total population. Due to differences in customs and living environments, there is a certain heterogeneity in the dietary structures of these different ethnic groups . MoreoveThe plant-based diet index (PDI) was also introduced in this study. We scored the PDI based on the frequency of intake. Furthermore, studies have shown that the use of non-quantitative food frequency questionnaires to assess dietary patterns is reliable and valid ,19. We ap < 0.05 were considered statistically significant. Statistical analyses were performed using IBM SPSS (version 26.0). The bar chart was produced by R-Package GGplot2 and the software version was 4.1.4.The frequency of detection and the concentration percentile of antibiotics were calculated based on demographic characteristics. Antibiotic concentrations below the LOD were replaced by concentrations at LOD/2 . The conp = 0.005) and detection concentration (p = 0.005) among the study subjects in different age groups were statistically significant (p = 0.02). The per capita monthly expenditure was not significantly different between the different population subgroups in terms of total detection rate and detected concentration. We also investigated the source of drinking water, frequency of eating eggs, frequency of drinking milk and goat’s milk, frequency of eating beef, frequency of eating mutton, frequency of eating seafood, frequency of eating vegetables, and frequency of eating fruits. The above factors did not show statistically significant differences in the total detection rate and detected concentration among the different population subgroups (p = 0.04) and detected concentration (p = 0.007) between the two groups of subjects who consumed pork and those who did not consume pork. There was a significant difference in antibiotic total detection rate (p = 0.02) and concentration (p = 0.03) in the population grouped by the PDI, and the highest detected antibiotic concentration (2500 ng/mL) was found in the population grouped by PDI in Q2, with the concentration of antibiotics showing a decreasing trend from Q2 to Q4. There was no significant difference in antibiotic total detection rate and concentration in the population grouped by uPDI, but it is noteworthy that the population grouped by uPDI showed an increasing trend in detected antibiotic concentration from Q2 to Q4.A total of 873 subjects participated in the study and the overall detection rate of urinary antibiotics was 49.8% . The difnificant . The fouubgroups . There wAccording to the classification of antibiotic preference, PVAs were detected at the highest rate (40.5%), followed by HAs (12.3%) and VAs (10.3%). PVAs were detected at the highest concentration (51 ng/mL), followed by HAs (3.1 ng/mL) and VAs (2.2 ng/mL). Moreover, the detection rate of PVAs was significantly different between the three groups who did not eat beef, occasionally ate beef, and ate beef every day, respectively, the two groups who ate fruit every day and did not eat fruit every day, respectively, and between people of different ages and people with different levels of education. The detection rate of VAs was significantly different between the two groups who ate eggs 1–3 times per month and those who ate eggs 1–3 times per week.p = 0.02) and concentration (p = 0.002) of thiamphenicol between the pork-eating and non-pork-eating groups, as well as a statistically significant difference in the concentration of sulfadiazine between the two groups (p = 0.01). Oxytetracycline and norfloxacin were specific in those who ate fruit daily. There was a significant difference in the detected concentrations of doxycycline between those who ate vegetables daily and those who did not eat vegetables daily. Differences were found in the detection rates and concentrations of tetracyclines, ciprofloxacin, ofloxacin, and chloramphenicol in the PDI group. In addition, there was a statistically significant difference in the concentrations of fluoroquinolones and sulfamethoxazole in the PDI group. In contrast, there was specificity in the uPDI group with respect to both the detection rate and the detected concentration of ofloxacin. The detection rates and concentrations of fluoroquinolones and norfloxacin were generally higher in those with low education than in those with high education. There were also significant differences in the detection rates and concentrations of fluoroquinolones and norfloxacin among different age groups.The presence of at least two antibiotics in the body was detected in 20.4% of the study subjects. Individual antibiotic detection rates ranged from 0.1% to 19.2% . OfloxacOR = 15, 95% CI = 1.7–131). Daily fruit eaters were more likely to have norfloxacin detected  compared to those who did not eat fruit daily, and the levels of norfloxacin were statistically significant in the low concentration group (Tier 2) and high concentration group (Tier 3) compared to the below the LOD group (Tier 1) for those who ate fruit daily. In the population with a PDI, the Q3 group was significantly associated with chloramphenicol , and the Q4 group was significantly associated with fluoroquinolones  and chloramphenicol . In addition, the Q2 group was statistically significantly associated with high fluoroquinolone concentration levels (Tier 3) compared to those in the group below the LOD (Tier 1).Univariate variables that were statistically significant were included in a multifactorial analysis to explore independent correlations in the detection rate and concentrations of antibiotics in human urine, and factors influencing the occurrence of urinary antibiotics . OccasioWith the aim of preventing antibiotic resistance, our research concerned the impact of antibiotics on human health. This study investigated urinary antibiotic exposure and potential determinants of antibiotic exposure in adults in Xinjiang. The detected concentrations and detection rates of 18 antibiotics were measured and compared in different adult groups. Similar to previous reports, we observed a large variation in the detection rates of different antibiotics ,23,24. TIn this study, at least two antibiotic residues were detected in 20.4% of the population, indicating widespread exposure of adults in Xinjiang to multiple environmental antibiotics. Despite the potential human health effects of exposure to antibiotics in the daily environment , data from comprehensive biomonitoring-based investigations of human antibiotic exposure remain limited. In fact, to date, human antibiotic exposure has only been reported in China and Korea ,25,26,27In addition, we detected samples of HAs that are not commonly used as veterinary drugs . These HAs have also been detected in adults in other parts of China  ,23. HoweE. coli in pigs from large pig farms in several Chinese provinces showed very high antibiotic detection rates, especially in Henan and Hubei provinces [Dietary intake is considered to be one of the important routes of chronic low-dose exposure to antibiotics in humans ,25. Consrovinces . This surovinces . Our sturovinces . Consistrovinces . The rearovinces . In addirovinces . In addirovinces . ConsistThis biomonitoring-based study reflected the level of antibiotic exposure among adults living in Xinjiang. None of the participants reported using any of the antibiotics analyzed within one month of the study, ruling out interference of iatrogenic antibiotics. However, there are some limitations in this study. First, antibiotic exposure in adults may be underestimated as some other common antibiotics, such as β-lactams and other types of tetracycline, were not analyzed in this study. However, these antibiotics have been detected in water environments in China and European countries in previous studies, and human exposure to environmental antibiotics may be higher ,43. HoweThe current study showed that adults in Xinjiang are widely exposed to a variety of antibiotics. Fluoroquinolones were found to be the most important antibiotics in terms of exposure, followed by tetracyclines. Pork and fruit intake may be potential sources of antibiotic exposure. Further research is needed to investigate the health effects of antibiotics in humans.


To describe acute respiratory illnesses (ARI) visits and antibiotic prescriptions in 2011 and 2018 across outpatient settings to evaluate progress in reducing unnecessary antibiotic prescribing for ARIs.Cross-sectional study.Outpatient medical and pharmacy claims captured in the IBM MarketScan commercial database, a national convenience sample of privately insured individuals aged <65 years.International Classification of Diseases, Tenth Revision, Clinical Modification (ICD-10-CM) codes.We calculated the annual number of ARI visits and visits with oral antibiotic prescriptions per 1,000 enrollees overall and by age category, sex, and setting in 2011 and 2018. We compared these and calculated prevalence rate ratios (PRRs). We adapted existing tiered-diagnosis methodology for In our study population, there were 829 ARI visits per 1,000 enrollees in 2011 compared with 760 ARI visits per 1,000 enrollees in 2018. In 2011, 39.3% of ARI visits were associated with ≥1 oral antibiotic prescription versus 36.2% in 2018. In 2018 compared with 2011, overall ARI visits decreased 8% , whereas visits with antibiotic prescriptions decreased 16% . Visits for antibiotic-inappropriate ARIs decreased by 9% , and visits with antibiotic prescriptions for these conditions decreased by 32%  from 2011 to 2018.Both the rate of antibiotic prescriptions per 1,000 enrollees and the percentage of visits with antibiotic prescriptions decreased modestly from 2011 to 2018 in our study population. These decreases were greatest for antibiotic-inappropriate ARIs; however, additional reductions in inappropriate antibiotic prescribing are needed. Inappropriate prescribing is even higher for acute respiratory illnesses (ARIs), for which about half of prescriptions are unnecessary.Antibiotics are common outpatient medications, However, few studies have examined temporal trends in unnecessary prescribing across outpatient settings for this period. This may be due to challenges created by diagnostic code changes in 2015 when the International Classification of Diseases, Tenth Revision, Clinical Modification (ICD-10-CM) replaced ICD-9-CM. Differences between ICD-9-CM and ICD-10-CM codes may complicate comparisons before and after the switch, although codes for ARIs may correspond better than for other diagnoses. Recent studies have described unnecessary antibiotic prescribing before (2010–2015) or after (2016) ICD-10-CM implementation. Additionally, temporal trends in unnecessary prescribing for ARIs have not been assessed in urgent care and retail health settings.From 2011 to 2018, overall US outpatient antibiotic prescriptions decreased 5%.The primary objective of our study was to describe ARI visits and antibiotic prescriptions in 2011 and 2018 across outpatient settings to evaluate progress in reducing unnecessary antibiotic prescribing for ARIs. and has previously been used to describe US outpatient antibiotic prescribing.We identified outpatient visits and oral antibiotic prescriptions in the 2011 and 2018 IBM® MarketScan® Commercial Database  based on median age during enrollment each year. Region  was considered missing if multiple values were present in a year. We categorized setting as office visits, outpatient hospital, urgent care, emergency department, retail health clinic, and other. If claims from multiple settings were present on the same date, we categorized setting as multiple. Because this was a visit-based analysis, enrollees could contribute >1 visit per year.We identified antibiotics using MarketScan therapeutic class designations: aminoglycosides, β-lactam antibiotics, cephalosporins, chloramphenicol, erythromycin and macrolides, miscellaneous antibiotics, penicillins, quinolones, sulfonamides , tetracyclines, and urinary anti-infectives. We additionally included metronidazole and trimethoprim. We included oral antibiotic prescriptions with ≥1 day supply and excluded those marked as refills. If multiple antibiotic classes or agents were dispensed on the same day, we categorized the class or agent as multiple.We assigned antibiotic prescriptions to the most recent outpatient visit within a 4-day window (the visit date and the subsequent 3 days). As the antibiotic indication could only be assumed based on diagnoses assigned at visits, we excluded prescriptions not linkable to a visit . Prescriptions linked to hospital discharges were excluded. Visits associated with multiple prescriptions dispensed on different days within the 4-day post-visit window were counted only once, and antibiotic class was categorized based on the first prescription dispensed. for ICD-10-CM codes. ICD-10-CM codes were categorized by condition and tier by author consensus. This system assigns a single diagnosis category per visit based on the diagnosis most likely to result in an antibiotic prescription (see Supplementary Reference Files). The system divides conditions based on systemic antibiotic indication. Tier 1 contains conditions for which antibiotics are almost always indicated . Tier 2 contains conditions for which antibiotics are sometimes indicated . Tier 3 contains conditions for which antibiotics are almost never indicated  and other conditions. We categorized bronchitis, bronchiolitis and asthma, allergy visits with additional diagnostic codes for chronic bronchitis , emphysema , or chronic obstructive pulmonary disease  as other respiratory conditions. Unlike the original framework, we categorized all codes previously captured as viral pneumonia (tier 3) as pneumonia (tier 1) because viral pneumonia codes were rarely used in outpatient settings. We additionally added 2 new categories: acute exacerbation of COPD (tier 2) and fever (tier 3) to characterize antibiotic prescribing for these conditions. To capture only antibiotic prescribing for fever with no other conditions, visits with tier 3 diagnostic codes in addition to fever were categorized as all other codes not listed elsewhere. For consistency, these changes were made for both ICD-9-CM and ICD-10-CM codes and, where appropriate, ICD-9-CM code categorizations were updated to mirror the ICD-10-CM classifications. We excluded encounter codes . Final diagnosis and tier were assigned to each visit based on the first-listed, lowest-tier condition. We applied the tiered-diagnosis algorithm to all visits in our study population. Only visits with ARI diagnoses  from the tiered-diagnosis algorithm were included for further analysis. We limited our study to ARIs to understand visit and antibiotic prescribing changes related to factors other than diagnostic coding.We adapted the ICD-9-CM antibiotic-indication tiered-diagnosis system from Fleming-Dutra et alWe calculated the annual number of ARI visits and ARI visits with oral antibiotic prescriptions per 1,000 enrollees overall and by age category, sex, and setting in 2011 and 2018 in our sample. The number of enrollees was calculated by weighting each enrollee based on their enrollment time during the year. For example, enrollees covered for the entire year were weighted as 1 and those covered for 6 months were weighted as 0.5.2 tests of proportion. We calculated prevalence rate ratios (PRRs) and CIs comparing the number of ARI visits and antibiotic prescriptions per 1,000 enrollees in 2011 versus 2018 , using a Poisson distribution overall and for each strata. Due to the large sample size in our study, we used an α of .0001, similar to previous studies using these data. All analyses were conducted using SAS version 9.4 software .We additionally calculated the percentage of ARI visits with antibiotic prescriptions and used a binomial distribution to estimate corresponding confidence intervals (CIs). We compared the percentage of ARI visits with antibiotic prescriptions in 2011 and 2018 using χThese data have been determined to be non–human-subjects research; thus, this study was not subject to institutional review board review by the National Center for Emerging and Zoonotic Infectious Diseases human-subjects advisor.In 2011 and 2018 combined, there were 392,619,001 total outpatient visits captured in the MarketScan commercial database that met our inclusion criteria. 11% of these  were associated with ARI diagnoses and were included in our final study population. In 2011, there were 829 ARI visits per 1,000 enrollees, whereas in 2018, there were 760 ARI visits per 1,000 enrollees  of ARI visits were associated with ≥1 oral antibiotic prescription versus 36.2%  in 2018 (Table 1). From 2011 to 2018, the percentage of ARI visits with antibiotic prescriptions decreased by 4.5  percentage points in children and 2.3 percentage points in adults.Children 0–17 years) accounted for 40% of ARI visits in 2011 and 37% of ARI visits in 2018 (Table 7 years aAntibiotic-inappropriate ARIs (tier 3) accounted for >50% of all ARI visits in 2011 and 2018 (Table ARIs for which antibiotics are sometimes indicated tier 2) accounted for majority visits with antibiotic prescriptions in our sample, and prescribing for these conditions decreased 9%  from 2011 to 2018 , and the percentage of antibiotic-inappropriate ARI visits with antibiotic prescriptions decreased from 20.9%  in 2011 to 15.5%  in 2018.Antibiotic prescribing per 1,000 enrollees decreased for all conditions except pneumonia and influenza. However, the percentage of influenza visits with antibiotic prescriptions decreased from 15.4%  in 2011 to 8.2%  in 2018. The greatest decreases in per-enrollee antibiotic prescriptions were observed for asthma and allergy and bronchitis and bronchiolitis, conditions for which antibiotics are almost never indicated. Nonetheless, in 2018 >60% of bronchitis and bronchiolitis visits were still associated with antibiotic prescriptions.1).Stratified by age, the conditions accounting for the highest proportions of ARI visits with oral outpatient antibiotic prescriptions were sinusitis, pharyngitis, bronchitis, and viral URI in adults and AOM, pharyngitis, and sinusitis in children (Supplementary Table 2). During the study period, the percentage of ARI visits with antibiotic prescriptions decreased across settings except in the other settings category, driven by lower prescribing for antibiotic-inappropriate ARIs across all settings . From 2011 to 2018, prescriptions decreased across all classes except penicillins, tetracyclines, and other. Decreases were especially notable among fluoroquinolones , sulfonamides , and macrolides . Among antibiotic-inappropriate ARIs (tier 3), macrolides were the most common agents, accounting for >50% of all prescriptions in 2011 and 2018. However, between 2011 and 2018, the number of macrolide prescriptions per 1,000 population for these conditions decreased 38% .Penicillins, macrolides, and cephalosporins accounted for the highest proportion of antibiotic prescriptions by class (Supplementary Table 4).By region, the South accounted for the largest proportion of visits in both 2011 and 2018 , suggesting a decrease in unnecessary antibiotic prescribing. However, in 2018 antibiotics were still prescribed in >15% of visits for antibiotic-inappropriate ARIs. Antibiotic prescriptions for ARIs decreased across outpatient settings, and the percentage of visits with antibiotic prescriptions was highest in urgent care facilities compared with other settings in both 2011 and 2018. and non-antibiotic care for some ARIs. Some of the observed decreases are likely related to decreased ARI incidence from uptake of the 13-valent pneumococcal conjugate vaccine, introduced in 2010. Some decreases may also be related to shifts in care to settings not captured in medical claims from which the MarketScan data are sourced . Notably, we observed an increase in influenza visits, likely related to greater influenza incidence in the 2017–2018 and 2018–2019 flu seasons compared with 2010–2011 and 2011–2012.There are likely multiple reasons for fewer ARI visits in 2018 compared with 2011. Care-seeking behaviors for ARIs may have shifted during the study period, potentially related to public health campaigns and previous exposures to delayed prescribing protocols and sinusitis in 2012–2013. Observed decreases in AOM and sinusitis visits with concurrent increases in viral URI suggest that cases that may previously have been diagnosed as AOM or sinusitis but not treated with antibiotics may have been assigned viral URI diagnoses instead.Conditions for which antibiotics are sometimes appropriate (tier 2) accounted for most visits with antibiotic prescriptions. Among these conditions, the percentage of visits with antibiotic prescriptions decreased only for pharyngitis. Notably, the percentage of pediatric sinusitis and AOM visits with antibiotic prescriptions increased during the study period. Some of the increased proportion of sinusitis and AOM visits with antibiotic prescriptions may be related to the implementation of more stringent diagnostic criteria for AOM However, opportunities to reduce antibiotic prescribing for antibiotic-inappropriate ARIs remain. For example, although antibiotic prescribing for bronchitis and bronchiolitis decreased during the study period, in 2018, >60% of visits for these conditions were still associated with antibiotic prescriptions.The greatest decrease in antibiotic prescribing occurred in visits for antibiotic-inappropriate (tier 3) ARIs. The decrease in all ARI visits and antibiotic prescriptions suggests that decreases in prescriptions for antibiotic-inappropriate ARIs reflect true improvements in prescribing rather than just changes in diagnostic patterns. This decrease is at least partially due to improvements in reducing unnecessary antibiotic prescriptions, potentially the result of outpatient antibiotic stewardship efforts, such as the Centers for Disease Control and Prevention Core Elements for Outpatient Antibiotic Stewardship. Shifts in outpatient care setting might explain discrepancies observed in visit trends between our study and a national study including only physician offices and EDs: the study of visits to physician offices and EDs found a 25% decrease in ARI visits from 2010–2011 to 2014–2015, compared with only an 8% decrease in ARI visits in our study population from 2011 to 2018.By setting, physicians’ offices accounted for >80% of ARI-related visits in both 2011 and 2018. Even so, we observed an increase in visits to emergency departments, urgent care facilities, and retail health clinics in 2018 compared with 2011. This shift is likely related to the changing landscape of outpatient healthcare with more low-acuity, outpatient visits presenting to urgent care and retail health settings. Although inappropriate prescribing was highest in urgent care settings for these conditions, we observed a marked decrease in unnecessary prescribing for antibiotic-inappropriate ARI visits, from 50% in 2011 to 35% in 2018, potentially reflecting the success of antibiotic stewardship efforts in urgent care in recent years.In both 2011 and 2018, the highest proportion of visits with antibiotic prescriptions for antibiotic-inappropriate (tier 3) ARIs was in urgent care facilities, congruent with a previous study using these same data. Differential improvement in prescribing to children may be a result of targeted stewardship efforts in this population, pneumococcal vaccination, and a culture of prescribing improvement among pediatricians. Decreased fluoroquinolone and macrolide prescribing likely reflects greater awareness of the potential harms of these drugs. Despite decreases in macrolides, azithromycin remained the most commonly prescribed antibiotic for antibiotic-inappropriate ARIs.We observed greater decreases in antibiotic prescribing to children compared with adults and marked decreases in fluoroquinolone and macrolide prescribing for ARIs, mirroring national antibiotic prescription trends. A strength of this study was the development and implementation of an antibiotic-indication tiered-diagnosis scheme for ICD-10-CM codes in a large sample capturing visits across outpatient settings.Our study had several limitations. First, our study was conducted in a convenience sample of commercially insured individuals aged <65 years and may not be generalizable to other populations. Second, our analysis used diagnostic codes from claims to assign a single diagnosis to each visit and we had no additional clinical information. Third, diagnostic codes and classifications vary between ICD-9-CM to ICD-10-CM; therefore, some changes observed in our study may reflect coding changes versus true shifts in ARI visits and antibiotic prescribing. However, codes for ARIs may correspond better between the 2 schemes  than for other conditions, so coding changes are likely minor factors in the overall ARI trends observed in our study. Fourth, our visit–prescription linking methodology may have misattributed some prescriptions to visits. Finally, we only examined 2 years (2011 and 2018), so we could not evaluate trends that occurred between those years. However, national outpatient antibiotic prescriptions showed consistent decreases during this period. are, at least in part, due to reductions in inappropriate prescribing, potentially reflecting stewardship efforts. Although reductions in inappropriate prescribing are encouraging, the decrease observed from 2011 to 2018 was modest. In 2018 inappropriate antibiotic prescriptions still accounted for 63 prescriptions per 1,000 enrollees. Additional focus on outpatient antibiotic stewardship is needed to improve outpatient antibiotic prescribing for ARIs. The ICD-10-CM tiered-diagnosis categorization developed for this study may assist in future efforts to track and improve outpatient antibiotic use.In conclusion, in this study of >40 million ARI visits using adapted tiered-diagnosis schemes for both ICD-9-CM and ICD-10-CM, we observed a decrease in overall antibiotic prescribing for ARIs driven by decreases in prescribing for antibiotic-inappropriate ARIs and pharyngitis in 2018 compared with 2011. Our findings support the hypothesis that observed decreases in antibiotic prescriptions nationally during this period


Dear Editor,1 Although CaOx nephrocalcinosis is usually asymptomatic, it can also induce severe inflammation and necrosis of renal tubular epithelial cells (TECs). Necrotic TECs release various damage-associated molecular patterns (DAMPs), which induce renal necroinflammation by activating Toll-like receptor 4 (TLR4) and secreting IL-1β, which triggers proinflammatory M1 macrophages (M1Mϕs) and then amplifies CaOx-induced renal injury,2 which can induce further crystal deposition and eventually lead to end-stage renal disease.3 However, information on the pharmacotherapeutics of kidney crystal formation remains limited. In addition, numerous studies have shown that CaOx crystal-induced oxidative stress and inflammatory injury to TECs are vital factors in the progression of crystalline nephropathy.4 Therefore, putting renal inflammation under control is expected to alleviate CaOx-induced inflammatory responses and inflammation-related tubular cell injury.Kidney stones are one of the most common clinical diseases in urology. Approximately 80% of renal calculi are composed of calcium oxalate (CaOx) crystals, which are gradually deposited in the medullary collecting duct or in the renal interstitium, causing nephrocalcinosis.5 Sirtuin1 (Sirt1), an NAD+-dependent protein deacetylase, is significantly activated by Met-mediated promotion of NAD+ and the NAD+/NADH ratio and participates in various cellular processes, such as suppressing inflammation, enhancing M2Mϕ polarization, reducing oxidative stress and maintaining mitochondrial function. Determination of whether Met can systemically tune down exaggerated immune responses via inflammatory signaling cascades and can be applied as a therapeutic agent to treat CaOx-induced kidney injury is urgently needed.In principle, the inflammatory responses induced by CaOx crystals involve diverse downstream effectors. Therefore, targeting particular cytokines or even signaling pathways may not be sufficient to alleviate kidney injury. Metformin (Met) is a pleiotropic drug that functions mainly by targeting oxidative stress and inflammation. It has been reported to protect renal tubular cells from inflammation, apoptosis, reactive oxygen stress, and endoplasmic reticulum stress in acute or chronic kidney diseases.In this study, we sought to understand the underlying pathways regulated by Sirt1 and explored the mechanism by which Met regulates CaOx nephrocalcinosis. We pretreated mice with increasing doses of Met  for 3 days, after which a model of CaOx nephrocalcinosis was established for 7 days . Reduced CaOx crystal deposition was observed when the Met concentration increased on day 10. Importantly, Met attenuated kidney injury in TECs caused by CaOx nephrocalcinosis in a dose-dependent manner Fig. . Moreove2FC |≥ 1.5; P value < 0.05). Gene Ontology enrichment and differential gene expression confirmed that the presence of CaOx crystals increased a spectrum of proinflammatory responses , macrophages recruitment and polarization  . The matrix dot plot enrichment of eight immune cell types showed a significant increase in renal macrophages infiltration in the Gly group  on glyoxalate (Gly)- or saline-treated mouse kidneys (GSE192703). Gly-induced CaOx nephrocalcinosis caused the upregulation of 2596 genes and the downregulation of 2161 genes (|logR4) Fig. . To furtoup Fig. , indicatGiven the anti-inflammatory effect of Met and its known regulatory effects on macrophages, we next performed RNA-seq on calcium oxalate monohydrate (COM)-stimulated bone marrow-derived macrophages (BMDMs) (GSE192593) pretreated with Met or PBS in TECs or COM-TECs . Analysis of differential gene expression confirmed that COM-TECs led to upregulation of a spectrum of proinflammatory transcripts in BMDMs Fig. . KEGG pafl/fl:Lysm-Cre mice and COM-TECs from WT mice. As expected, Sirt1 knockout (KO) significantly promoted M1Mϕs polarization and increased TLR4 signaling (Fig. We hypothesized that Met exerts its anti-inflammatory effects partly by interfering with TLR4 and IL-1β through Sirt1 in macrophages and modulating macrophages polarization. To evaluate this hypothesis, we cocultured COM-stimulated TECs with Met-treated BMDMs. Fig. ing Fig.  Moreovering Fig. . The resfl/fl:Lysm-Cre mice with Met for 3 days and developed a CaOx nephrocalcinosis mouse model by the intraperitoneal injection of glyoxylic acid. We determined that Met significantly reduced kidney CaOx crystal deposition in the WT mice, while Sirt1cKO reversed this effect (Fig. 18F-FDG uptake by micro-PET-CT to determine the local renal inflammatory response. Micro-PET-CT imaging revealed that Met decreased 18F-FDG uptake in the WT mouse kidneys, while these parameters were enhanced in the Sirt1fl/fl:Lysm-Cre mice (Fig. We further explored whether the effects of Met on CaOx-induced renal inflammation and crystal deposition are Sirt1dependent. We pretreated the WT and Sirt1ect Fig. . We analice Fig. . Furtherice Fig. . PAS, TUice Fig. . In addiice Fig. . In summIn conclusion, the results demonstrated that Met can attenuate crystal deposition and kidney injury in a CaOx nephrocalcinosis mouse model by promoting Sirt1 activation and M2Mϕs polarization. A combination of Met with certain anti-oxalate drugs may provide a more effective treatment and prevent recurrence in CaOx nephrocalcinosis. Our study provides strong evidence supporting the therapeutic and preventative potential of using Met in clinical settings.Supplemental Material


The clinicopathological phenotype of G2019S LRRK2‐associated Parkinson's disease (L2PD) is similar to idiopathic Parkinson's disease (iPD), and G2019S LRRK2 nonmanifesting carriers (L2NMCs) are at increased risk for development of PD. With various therapeutic strategies in the clinical and preclinical pipeline, there is an urgent need to identify biomarkers that can aid early diagnosis and patient enrichment for ongoing and future LRRK2‐targeted trials.The objective of this work was to investigate differential protein and phospho‐protein changes related to G2019S mutant LRRK2 in peripheral blood mononuclear cells from G2019S L2PD patients and G2019S L2NMCs, identify specific phospho‐protein changes associated with the G2019S mutation and with disease status, and compare findings with patients with iPD.We performed an unbiased phospho‐proteomic study by isobaric label–based mass spectrometry using peripheral blood mononuclear cell group pools from a LRRK2 cohort from Spain encompassing patients with G2019S L2PD (n = 20), G2019S L2NMCs (n = 20), healthy control subjects (n = 30), patients with iPD (n = 15), patients with R1441G L2PD (n = 5), and R1441G L2NMCs (n = 3) .Comparing G2019S carriers with healthy controls, we identified phospho‐protein changes associated with the G2019S mutation. Moreover, we uncovered a specific G2019S phospho‐signature that changes with disease status and can discriminate patients with G2019S L2PD, G2019S L2NMCs, and healthy controls. Although patients with iPD showed a differential phospho‐proteomic profile, biological enrichment analyses revealed similar changes in deregulated pathways across the three groups.Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder SocietyWe found a differential phospho‐signature associated with LRRK2 G2019S for which, consistent with disease status, the phospho‐profile from PD at‐risk G2019S L2NMCs was more similar to healthy controls than patients with G2019S L2PD with the manifested disease. © 2022 The Authors.   LRRK2) gene cause autosomal‐dominant LRRK2‐associated Parkinson's disease (L2PD) with a clinical phenotype similar to idiopathic Parkinson's disease (iPD).Mutations in the leucine‐rich repeat kinase 2  domain enhance LRRK2 activity,Our study aimed to explore protein and phospho‐protein systemic changes related to mutant LRRK2 using accessible blood cells from G2019S carriers and to identify the functional pathways associated with G2019S. Supported by The Michael J. Fox Foundation for Parkinson's Research, we established the Barcelona LRRK2 Biorepository, a collection of peripheral blood mononuclear cells (PBMCs) from G2019S mutation carriers along with detailed clinical data on each enrolled participant. Using this cohort, we performed an unbiased phospho‐proteomic discovery study by isobaric label–based mass spectrometry (MS). In Alzheimer's disease (AD), phospho‐proteome approaches have identified signaling pathways altered during early disease progression,l‐dopa) equivalent daily dose than patients with iPD (695 vs. 672 mg) and a higher proportion of patients taking l‐dopa (88% vs. 73%). The higher percentage of l‐dopa–treated L2PD patients can be related to their slightly longer evolution time (8.4 ± 4.3 vs. 7.3 ± 3.4 years) and their relatively worse Schwab & England daily activity of 91% (70–100) versus 97% (80–100). Overall, we did not find significant medication differences between L2PD and iPD. The local ethics committee of the Hospital Clínic de Barcelona approved the study, and all subjects gave written informed consent.Blood samples from G2019S and R1441G mutation carriers (patients with L2PD and L2NMCs), patients with iPD, and healthy controls were obtained at the Movement Disorders Unit from the Hospital Clínic de Barcelona. Patients with PD were clinically diagnosed according to the UK PD Society Brain Bank criteria, except that more than one affected relative with PD was not an exclusion criterion.We used TaqMan SNP assays‐on‐demand on a Step One Plus Real‐time PCR System  to genotype LRRK2 G2019S  and a commercial TaqMan assay for R1441G/C/H as previously described.2 and stored at −80°C until use. The average time between blood collection and flash‐freeze of dry pellets was 4 h, and the average storage period at −80°C was less than 1 year.A total of 8 mL of blood was drawn by peripheral vein puncture in fasting. PBMCs were isolated by density gradient using sodium‐citrate collector tubes  according to the manufacturer's instructions. Dry pellets were flash‐frozen in liquid Ng for 1 hour at 15°C. After precipitation, protein concentration was measured in the supernatants using the Bradford assay kit . Reduction, alkylation, digestion, and tandem mass‐tag labeling were performed as described in the Supporting Information Methods  and phosphatase inhibitors . Homogenates were spun down at 100,000Appendix .Enrichment of phosphorylated peptides was performed applying the SIMAC  protocol as previously described.H. sapiens . The parameters used were as follows: initial maximum precursor (25 ppm), fragment mass deviations (40 ppm), variable modification  and fixed modification , enzyme (trypsin) with a maximum of 1 missed cleavage, minimum peptide length (7 amino acids), false discovery rate (FDR) for peptide spectrum matches (PSM) and protein identification (1%). Frequently observed laboratory contaminants were removed. More specifically, during protein sample isolation and processing, potential contaminants encompass all subtypes and isoforms of keratins, hair keratins, keratin‐like proteins, and cytoskeletal keratins, which can be ubiquitously present in the skin, dust, clothes, and chemicals, among others. To avoid any potential interference with the PBMC proteomes, we excluded all these contaminants during protein identification, quantitation, and database search phases. To that end, we used the contaminants.fasta file from the Maxquant installation package, which contains the primary sequence data of conventional proteases used in proteomics experiments and all human keratin subtypes. Protein identification was considered valid with at least one unique or “razor” peptide. Protein quantification was calculated using at least two razors + unique peptides. Statistical significance was calculated by a 2‐tailed Student t test (P < 0.05) in pairwise comparisons and by one‐way ANOVA (P < 0.05). For the DPPs, in the analysis of the PBMC phosphorylated fractions, phospho‐serine, phospho‐threonine, and phospho‐tyrosine were chosen as variable modifications for database searching. We used the Perseus software (version 1.5.6.0)Raw MS/MS spectra were processed using the MaxQuant software (version 1.5.8.3)P < 0.01). As a statistical significance cutoff, we used a false discovery rate multiple testing adjusted P < 0.05. We also performed a network analysis of the interlocked DEPs and DPPs identified in patients with G2019S L2PD, G2019S L2NMCs, and patients with iPD in Ingenuity Pathway Analysis v62089861  (www.ingenuity.com)To elucidate the biological functions of DEPs and DPPs identified in patients with G2019S L2PD, G2019S L2NMCs, and patients with iPD, we performed a biological enrichment analysis in Metascapet test (P < 0.05). Comparing patients with G2019S L2PD with healthy control subjects, we found 100 DEPs and 37 differential phospho‐peptides from 30 DPPs, owing to the fact that phosphorylation at a given protein can occur at one or more Ser/Thr/Tyr residues.By unbiased isobaric label‐based MS, we identified 2374 proteins expressed in human PBMCs Fig. . We firsP < 0.05). We found 94 DEPs among patients with G2019S L2PD, G2019S L2NMCs, and healthy controls , in iPD, we found 101 DEPs and 33 differential phospho‐sites from 26 DPPs with respect to healthy controls. We also found 85 DEPs and 35 phospho‐sites from 23 DPPs in iPD compared with patients with G2019S L2PD , which were earlier related to L2PD. RAB regulation of vesicle trafficking was also among the top terms in G2019S carriers. Alternatively, we analyzed the interlocked DEPs and DPPs independently in patients with G2019 L2PD, G2019S L2NMCs, and patients with iPD . Beyond common network deregulation in iPD, G2019S L2PD, and G2019S L2NMCs (centered around AKT and NFKB)  terms indicating overlapping functions . The protein differences in the G2019S L2PD and G2019S L2NMC groups independently were higher than in all G2019S carriers as a whole, thus suggesting additional specific protein phosphorylation differences in PD and non‐PD G2019S carriers. Between G2019S L2PD and G2019S L2NMCs, we found the largest protein phosphorylation differences across all pairwise comparisons (107 DEPs and 56 DPPs). A proteomic study in urine showed DEP changes from G2019S L2NMCs to G2019S L2PD patients.Second, as a proof of principle, by analysis of variance (ANOVA), we confirmed that the G2019S LRRK2 DPPs change with disease status from asymptomatic G2019S carriers to patients with L2PD with overt disease. Specifically, we uncovered a phospho‐protein signature of 32 phospho‐peptides from 23 unique DPPs with discriminative features to differentiate patients with G2019S L2PD, G2019S L2NMCs, and healthy controls. Consistent with disease status, asymptomatic G2019S L2NMCs were more similar to healthy controls than patients with G2019S L2PD. Because these observations were made in pooled PBMC samples from patients, we cannot establish correlations of DPPs and clinical parameters at the individual level at this time. However, our findings suggest a great potential for unbiased MS‐based approaches in analyzing individual samplesl‐dopa–induced dyskinesiasSYT11, which along with ATP13A2 regulates autophagy.The 23 DPPs of the differential phospho‐signature can be plausibly related to mutant LRRK2. Illustratively, MADD is a guanosine triphosphate/guanosine diphosphate exchange factor for RAB proteins.Third, we compared the PBMC phospho‐proteome of G2019S carriers and patients with iPD. The differential phospho‐signature was specific for G2019S carriers; however, the biological pathways affected by G2019S‐related DEPs and DPPs overlapped with iPD. These results suggest common disease mechanisms and possible common therapeutic approaches for L2PD and at least some iPD subtypes,Functionally, DEPs and DPPs at the G2019S phospho‐signature were involved in vesicle‐mediated transport, Rho GTPases, and phagocytosis, among other terms related to LRRK2 mutations.Interestingly, several of the DPPs identified in our study were also described in a phospho‐proteomic screening of PBMCs from healthy controls treated with the Lu AF58786 LRRK2 and PFE‐360 inhibitors (145 DPPs).5 and their phosphorylation status is rapidly dynamic.Despite some exciting observations on differences in signatures in LRRK2‐manifesting and nonmanifesting carriers, our study has limitations. We assessed posttranscriptional protein modification by phosphorylation mainly at Ser and Thr residues and Tyr residues, but not at noncanonical residues.In summary, we performed an unbiased phospho‐proteomic screening in PBMCs from a large G2019S LRRK2 cohort. We uncovered a differential phospho‐signature associated with the G2019S mutation, which changes between asymptomatic and symptomatic G2019S carriers. Compliant with disease status, asymptomatic G2019S carriers were more similar to healthy controls than patients with G2019S L2PD. By identifying phospho‐protein candidates related to LRRK2 G2019S, our study contributes to understanding the biological pathways of mutant LRRK2 and helps decipher the dynamic molecular changes occurring before and after phenoconversion. We also generated DEP and DPP datasets from G2019S carriers, which we provide publicly to other researchers for data mining and phospho‐signature validation. If validated at the individual level in future studies, our findings may hold implications for disease prediction, patient enrichment for clinical trials, and early PD detection in PD at‐risk G2019S carriers. Further MS‐based phospho‐proteomic studies in larger G2019S populations are warranted.E.T. received honoraria for consultancy from TEVA, Bial, Prevail, Boehringer Ingelheim, Roche, and BIOGEN.M.F. was funded by the María de Maeztu program (grant MDM‐2017‐0729 to the Parkinson's Disease and Movement Disorders group of the Institut de Neurociènices [Universitat de Barcelona]).R.F.‐S. was supported by a JIN grant of the MINECO and the AEI (AEI/FEDER/UE) (grant SAF2015‐73508‐JIN) and a Miguel Servet grant from the Instituto de Salud Carlos III (grant CP19/00048).R.F.‐S., M.E., C.M., and E.S. were supported by The Michael J. Fox Foundation for Parkinson's Research (MJFF) (grant MJFF‐000858).Conception and design of the study: Conception ; Organization ; Execution ; Patient recruitment .Acquisition and analysis of data: Design ; Execution ; Interpretation ; Review and Critique .Drafting of the manuscript or figures: Writing of the first draft (R.F.‐S.); Draft editing ; Review and Critique .Appendix S1. Supporting InformationClick here for additional data file.FIGURE S1. Volcano plot representations showing top DEPs and DPPs differences in comparisons involving G2019S L2PD and G2019S L2NMC, and controls based on fold‐change and P‐value criteria.A) Differentially expressed proteins (DEPs).(B) Differentially phosphorylated proteins (DPPs). and the largest (Manhattan) distance from the origin.https://huygens.science.uva.nl/VolcaNoseR/.VolcaNoSer software: Click here for additional data file.FIGURE S2. Comparison of DEPs and DPPs among iPD, G2019S L2PD, and G2019S L2NMC compared to healthy controls as evaluated by Student t test (P < 0.05).A) Differentially expressed proteins (DEPs).(B) Differentially phosphorylated proteins (DPPs). interlocked DEPs and DPPs.(B) DEPs and DPPs per separate. as analyzed using Metascape (Reactome database). This analysis showed similar pathway deregulation using interlocked DEPs and DPPs or DEPs and DPPs per separate in G2019S carriers.Statistically significant Gene Ontology (GO) terms were adjusted by multiple testing adjustment based MS of P‐values  AKT‐centred network in G2019S L2PD, G2019S L2NMC, and iPD.(B) NFKB‐centred network in G2019S L2PD, G2019S L2NMC, and iPD.(Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.Table S5Click here for additional data file.Table S6Click here for additional data file.Table S7Click here for additional data file.Table S8Click here for additional data file.Table S9Click here for additional data file.Table S10Click here for additional data file.


D-6 is synthesized and characterized. Host-guest interactions of D-6 with different ammonium guests produced a variety of fluorescence with hypsochromic shifts up to 130 nm, which are found to be affected by choice of solvent or guest and host/guest stoichiometry. Formation of supramolecular complexes were confirmed by UV-vis titration, 1H NMR and HRMS spectroscopy.Dynamic fluorophore 9,14-diphenyl-9,14-dihydrodibenzophenazine (DPAC) affords a new platform to produce diverse emission outputs. In this paper, a novel DPAC-containing crown ether macrocycle  Supramolecular chemistry  is underN,N′—diphenyl—dihydrodibenzo  phenazines (DPAC) possesses unique photophysical properties including the double fluorescence emission, large stokes shift and remarkable responsiveness to various environmental stimuli . In soluHerein, we designed and synthesized a large-size DPAC-based crown ether macrocycle D-6 whose dynamic DPAC chromophore was covalently locked by a conformational flexible hexaethylene glycol chain see ; 2) titr1H NMR, 13C NMR, and high-resolution mass spectrometry (HRMS) were used to confirm the chemical structure of D-6 , and the detection temperature was room temperature  unless otherwise specified. High resolution mass spectrometry (HRMS) was performed by Waters LCT Premier XE mass spectrometer, in which electrospray ionization (ESI) was used for ionization. The UV/Vis absorption spectra data were documented by a Shimadzu UV-2600 UV-Vis spectrophotometer and the fluorescent spectra were acquired by a Shimadzu RF6000 spectro fluorophotometer.The D-6 is not the first DPAC-involving crown ether we investigate. In a previous work of Qu group , G1 , G2 , G3 , G4 , and G5  , demonstrating the large impact of host-guest interactions. In particular, the addition of G5 to D-6 produced the largest shift of 130 nm from 584 nm (orange) to 454 nm (dark blue). The large variations of emission color in response to different guests were most likely caused by the conformational adaptation of D-6. The originally relaxed ethylene glycol backbone underwent a stronger resistance in tension after the insertion of the guests. Simultaneously, the wings of the DPAC unit were constrained to perform light-induced structural planarization, resulting in the changes of fluorescence. Potentially, the D-6 macrocycle could be used as a supramolecular fluorescent probe to distinguish different ammonium salts.Fluorescence spectra of the host-guest mixtures were then recorded at room temperature to examine the impact of supramolecular complexation on the fluorescence characteristics of 7, 0.19) . The emiG5 to D-6 was then carried out. As is clearly shown in G5, the main emission peak of D-6 at 584 nm gradually decreased while a peak around 470 nm arose and increased simultaneously. Eventually, the new peak stopped to increase when 1 eq. guest was added. Additionally, noticeable visual changes could be observed after each 0.1 equivalent G5 was added. The fluorescence spectra discussed above were also translated to CIE coordinates as following: 0 eq. , 0.1 eq. , 0.2 eq. , 0.3 eq. , 0.4 eq. , 0.5 eq. , 0.6 eq. , 0.7 eq. , 0.8 eq. , 0.9 eq. , 1.0 eq. . From the CIE chromaticity diagram . The spectra of D-6 (blue), G5 (red), and their equimolar mixture (green) were shown in G5 and D-6, the methylene proton Hf of G5 is observed to significantly upshifted by 0.48 ppm. Similarly, all the phenyl protons of G5 shifted to the higher field . Meanwhile, all of the protons of D-6 underwent displacements, especially H5,6, H9, and H14,15 . All the changes could be explained by the insertion of G5 into the cavity of D-6 and the consequent formation of the host-guest structure. Similar spectral variations were also found in the other four cases. In addition, the molecular ion peaks found for D-6 and its visitors in the HRMS data also support the complexation of them (The formation of the host-guest complexes was further confirmed by 400 MHz  of them .G1-G5 into the solutions of D-6) using nonlinear regression methods (D-6 along and with the five guests that the degrees of hypsochromic shifts increase in the order of G1-G5. Interestingly, the binding constants are perfectly in line with this sequence, showing the binding constant dependance of color change. The differences of binding constants were considered to be partially due to the different electron deficiency of the ammoniums. Comparing to G1-G3, the carbonyl groups of G4 and G5 increase the electron deficiency of their ammonium sites and consequently bring higher affinity in the complexation with the electron rich crown ether cavity. However, the relationship of the chemical inputs and the emission colors in the present work can hardly be contributed to only this reason. The long hexaethylene glycol backbone gives access to the guests with different sizes while the strong topological flexibility of the backbone gives possibility to the conformational adaptation of the DPAC unit and the whole macrocycle. Relationally, the different rigidities of the guests could also affect the binding geometry and the binding constants.By processing the spectra of UV-vis titrations (respective addition of  methods , a numbe methods . The larD-6 as a conformation-adaptive supramolecular host. By the respective incorporation of various ammonium guests, multicolor emission from orange to white to deep blue was accomplished. Solvent and host/guest stoichiometry were found to be effectors that influence the optical outputs. The supramolecular host-guest complexation was confirmed by UV-vis titration, 1H NMR, and HRMS data. The guest-dependent emission of D-6 shown in this work is potential to distinguish different ammoniums, which would be continuously studied by our group in future. The use of supramolecular chemistry to modulate emission wavelengths over a broad range afford an effective way to obtain multicolor emission within a less complicated system.In conclusion, relying on the light induced structural planarization of DPAC derivatives, we synthesized a new emissive macrocycle


To promote the in-situ and structural application of waste concrete in cold regions, the sustainable application potential of waste concrete in a freeze-thaw (F-T) environment was comprehensively evaluated from three aspects of performance, environmental load, and economic benefit. The recycled aggregate concrete (RAC) was produced by recycled coarse aggregate (RCA), which was obtained from the crushing of natural aggregate concrete (NAC) after every F-T 150 cycles until F-T failure. The effects of F-T damage of parent concrete on the physical properties of RCA and mechanical and frost resistance of RAC under 35% flexural stress were studied. Besides, the sustainability of NAC and RAC was compared and analyzed by emergy theory. The results suggested that the physical properties of RCA deteriorated gradually with the accumulation of F-T damage to parent concrete. The RCA obtained from parent concrete that suffered F-T damage could be used as coarse aggregate for structural concrete when F-T damage is smaller than 0.367. The F-T damage of parent concrete had an adverse effect on the mechanical properties and frost resistance of RAC. The frost resistance of RAC obtained from parent concrete with larger F-T damage was worse. The RAC prepared from parent concrete without F-T failure can serve 50 years in cold regions, while that with F-T failure can only serve 30 years. The F-T damage microelements were dispersed in the adhesive mortar of RCA and transferred to RAC, resulting in the reduction of the mechanical properties and frost resistance of RAC. Emergy analysis showed that the reuse of waste concrete after F-T failure required higher economic input, higher environment load, lower output efficiency, and sustainability. The performance, environmental load and economic benefit of RAC prepared by using waste concrete after F-T failure were inferior to that of waste concrete without F-T failure. Waste concrete after F-T failure is not recommended to be used as coarse aggregate for structural concrete. The adoption of sustainable consumption and production patterns is one of the key goals of the United Nations 2030 Agenda for Sustainable Development. At present, in order to achieve socially sustainable production and promote the development of a green economy, the recycling and utilization of materials have been paid attention to in many fields, such as medical treatment, food processing, bioenergy, electronic equipment, and construction ,2,3,4,5.The construction industry plays an important role in carbon emissions, and its sustainable production patterns are always being explored. Using waste concrete to produce recycled coarse aggregate (RCA) and reuse it in structural concrete can facilitate sustainable development and obtain remarkable economic effectiveness. Meanwhile, it is of great significance for the achievement of United Nations Sustainable Development Goals such as protecting terrestrial ecology, reducing carbon emissions, and coping with climate change. However, due to the different concrete service environments and cementitious materials compositions all over the world, the properties of RCA are different . The addIn the reusing process, natural aggregate concrete (NAC) is generally as the parent concrete. The compressive strength of parent concrete affects the content and quality of adhesive mortar of RCA , which lThe performance of parent concrete also has a significant influence on the mechanical properties and durability of recycled aggregate concrete (RAC) . Ahmad BIn cold regions, a large number of hydraulic structures are exposed to a freeze-thaw (F-T) environment, such as dams and bridges. The frost resistance of concrete becomes an important durability index to evaluate the health of structures. Once F-T destruction of concrete occurred, it will become waste concrete because of the loss of bearing capacity and protection ability to reinforcement . Due to At present, the frost resistance of RAC has been fully studied. The quality of RCA , replaceEmergy analysis is an effective method to evaluate the sustainability of renewable resource recovery systems . Based oThe purpose of this paper is to comprehensively evaluate the sustainable application potential of waste concrete in the F-T environment. C40 NAC was prepared as parent concrete. Under the simulated load-(F-T) coupling action, the parent concrete was crushed into RCA every F-T 150 cycles for the preparation of RAC until F-T failure. The effects of F-T damage on particle size and physical properties of RCA were analyzed. The F-T damage threshold of parent concrete was obtained based on the requirements of structural concrete aggregate. Besides, the effects of F-T damage of parent concrete on the mechanical properties and frost resistance of RAC were further studied, and the maximum service life of RAC in cold regions was obtained. Through the analysis of porosity and scanning electron microscope (SEM), the micro mechanism of the effect of parent concrete F-T damage on the properties of RCA and RAC was revealed. In addition, according to the emergy analysis theory, the emergy investment ratio (EIR), emergy yield ratio (EYR), environmental load ratio (ELR) and emergy sustainability index (ESI) of NAC and RAC were calculated, so that the sustainable utilization potential of waste concrete from three aspects of performance, environmental load and economic benefits were evaluated comprehensively.3 respectively. Besides, polycarboxylic acid (PCA) superplasticizer and GYQ-E100 air entraining agent (AEA) were also added to enhance the properties of concrete. The dosage of PCA and AEA is 0.5% and 0.06% of the quality of binding materials.Natural gravel was regarded as the natural coarse aggregate (NCA) to produce parent concrete. The physical properties of NCA were tested according to GB/T 14685(2011) , and thecf) test. Specimens with size of 100 × 100 × 400 mm were used for flexural strength (ff) test and rapid F-T test. The specimens were placed in the standard curing chamber (the temperature of 20 ± 2 °C and relative humidity > 95%) for 28 days. The slumps of fresh concrete were determined according to GB/T 50080(2016) [The strength grade of NAC was C40 and the target slump was 180 mm, which can satisfy the requirements of most projects. The ‘overall calculation method’  was used80(2016) , and thef). Under this stress condition, the microcracks are stable. The specific loading procedure is shown in The new rapid F-T test method of simulating load and F-T coupling test was adopted . The flecf and ff were tested every F-T 150 cycles.W0 represents the mass of concrete before F-T cycle; iW represents the mass of concrete after F-T i cycles.f0 represents the transversal fundamental frequency of concrete before F-T cycle; if represents the transversal fundamental frequency of concrete after F-T i cycles.The new method avoids stress loss due to the violent fluctuation of the rapid F-T test temperature. The mechanical properties and rapid F-T test of NAC were carried out according to GB/T 50081(2019)  and GB/TFive kinds of RCA from different F-T cycles of NAC were obtained. The NAC after 0, 150, 300, 450 and 600 F-T cycles were put into an oven with a temperature of 60 °C and dried for 24 h before crushing. To minimize the external load impact of crushing on RCA properties, NAC was crushed in jaw crusher twice. The outlet width of jaw crusher was set as 31.5 mm to control the particle size range (5–25 mm) of RCA. After cleaning and screening, RCA with particle size greater than 5 mm was obtained. The apparent density, water absorption, and crushing values of RCA were determined according to GB/T 25177(2010) . The adhFive kinds of RAC were prepared and the corresponding relationships among NAC, RCA and RAC are shown in cf, ff, MLR and RDME. The cf and ff were tested every 100 F-T cycles. The MLR and RDME were tested every 50 F-T cycles.After 28 days of curing, the rapid F-T test was conducted on RAC to access the frost resistance. The load and rapid F-T coupling test method were consistent with that of parent concrete. The frost resistance of RAC was evaluated in terms of the P is the porosity; m0 is the mass of RAC saturated by water absorption in vacuum; m1 is the constant mass of RAC under 105 °C; cρ and wρ are the densities of RAC and water respectively.The micromorphology of NAC and RAC after F-T cycles was observed with a JSM-IT100 scanning electronic microscopy instrument. The interfacial transition zone (ITZ) and pore changes of aggregate were observed. The change of porosity of RAC was measured by the ‘evaporable water content method’ . The forThree specimens were prepared for each group of concrete to obtain the average value of three data calculations as the group result, and calculate the standard deviation as the error. The experimental process is illustrated in E is emergy (sej); λ is emergy conversion rate (sej/J), which is the amount of emergy needed to produce one joule of product; aE is available energy (J).F is the emergy value of non-resource investment; Y is the emergy value of the product produced; N is the emergy value of non-renewable resources; R is the emergy value of renewable resources.Emergy analysis was used to analyze the sustainability of the RAC production mode. The emergy indexes involved in this study include: EIR, EYR, ELR and ESI. EIR reflected the level of economic input. The larger the EIR value of the production system, the higher the economic input required by the production system. EYR can reflect the output efficiency of the production system dynamically, and the larger the EYR value, the greater the production efficiency. ELR was used to evaluate the environmental burden caused by the production process. The more environmental burden caused, the greater the ELR value will be. Besides, ESI can evaluate the sustainability of production systems. The production system was sustainable and had great development potential when 1 ≤ ESI ≤ 10 . The calcf and ff of NAC were both decreased after F-T cycles. The cf of NAC-600 was 24.64% lower than that of NAC-0, while the ff was 43.37% lower than that of NAC-0. This indicated that the ff was more sensitive to F-T damage than cf. The strength decreased with the number of F-T cycles, which indicated more F-T damage produced in the NAC. This inevitably affects the physical properties of RCA after concrete crushing [The mechanical properties of NAC after F-T cycles are shown in The variations of MLR and RDME of NAC after F-T cycles are illustrated in n)( under different F-T cycles was obtained [Dn), the F-T damage Dobtained . The varobtained ,41,42. TFrost heave stress caused by F-T cycles repeatedly acted on the interior of NAC. When the stresses exceeded the tensile strength limit of NAC, cracks occurred in the internal structure of NAC, and cracks further expanded until F-T failure .i was obtained from the NAC every F-T 150 cycles until F-T failure. As the F-T cycles of NAC increased, the particle size proportion of 0–5 mm of RA increased and the proportion of RCA with particle size greater than 5 mm decreased.The particle size proportions of recycled aggregate (RA) are shown in RCAP represents the pick-up rate of RCA; RCAM represents the mass of RCA; RAM represents the mass of total RA.The pick-up rate of RCA is the ratio of the mass of RCA with a particle size above 5 mm to the mass of total RA, as shown in Formula (11). The pick-up rate of RCA was reduced with the development of F-T cycles of NAC. The pick-up rate of RCA1, RCA2, RCA3, RCA4 and RCA5 were 71.43%, 69.91%, 66.88%, 64.53% and 62.46% respectively. The pick-up rate of RCA5 was 12.56% lower than RCA1, which indicated that the F-T damage had a negative effect on the pick-up rate of RCA.The F-T damage resulted in the decrease of mechanical properties of NAC, which leads to more mortar peeling during the crushing process of NAC. With the increase of F-T cycles of NAC, the RCA particle size decreased and the number of mortar particles increased, which was bound to affect the physical properties of RCA .Pores and microcracks were generated in the F-T cycles of NAC and further expanded due to the external loads in the crushing process. It can be inherited from RCA, leading to the reduction of physical properties of RCA. Meanwhile, it can be learned from the previous Dn). GB/T 25177(2010)  specifieIn cf and ff of RAC-i. It can be found that the cf and ff of RACs decreased linearly with the increase of F-T cycles. In cf of RAC2 was 29.71% higher than that of RAC5, while the 28-d ff of RAC2 was 45.86% higher than that of RAC5, which indicated that the F-T damage of NAC had a more significant adverse impact on the ff of RAC.On the one hand, the F-T damage caused more holes and microcracks to appear in the adhesive mortar, weaken the ITZ, and had an adverse impact on the strength of RAC . On the i are shown in The variations of MLR and RDME of RAC-i are shown in The F-T damage of RAC was calculated according to formula (9). The variations of F-T damage of RAC-N represents the laboratory F-T cycles (n); y represents the average F-T cycles per year; t represents the service life (a) in a natural F-T environment.One rapid F-T cycle in the laboratory was equivalent to F-T 12 cycles in the natural environment . The conThe cold region was represented by North China, and its annual average F-T cycle was 84 cycles . AccordiIt can be found from The variations of the porosity of RAC are shown in Generally, as shown in The micro morphologies of NAC-0, NAC-300, and NAC-600 are illustrated in The micro morphology of RAC1, RAC3, and RAC5 after F-T 300 cycles are illustrated in The influence mechanism of NAC F-T damage on the mechanical and frost resistance of RAC is shown in 3 concrete. Due to space limitation, The emergy flow modes of NAC and RAC production are illustrated in The basic emergy flow and emergy evaluation index of concrete can be calculated according to The ESI of RACi was greater than 1, RACi production mode had better sustainability. Although the RACi production mode increased the investment of social resources, with the integration and upgrading of the construction waste treatment industrial chain , the graTo sum up, the performance, environmental load, and economic benefit of RAC prepared by using waste concrete after F-T failure are inferior to that of waste concrete without F-T failure. The waste concrete after F-T failure needed to be modified to improve RAC performance, which might cause a change in the emergy evaluation index. By developing a new RAC mix proportion, or further improving the modification technology of RCA and optimizing the proportion of various emergy inputs, the social resource input of RAC production can be reduced, the output efficiency of RAC can be enhanced, and then the sustainable application potential of waste concrete can be improved.In this paper, the effects of NAC freeze-thaw damage on the properties of RCA, the mechanical properties, and frost resistance of RAC were studied; besides, the sustainable utilization potential of waste NAC in a freeze-thaw environment was comprehensively evaluated from three aspects of performance, environmental load, and economic benefits. The specific conclusions are as follows.(1)As the number of freeze-thaw cycles of parent concrete increased, the particle size proportion of 0–5 mm of recycled aggregate increased and the pick-up rate of RCA reduced. The freeze-thaw damage of parent concrete adversely affected the physical properties of RCA. With the increase of freeze-thaw damage, the apparent density of RCA linearly decreased, while the crushing value and water absorption of RCA increased. The RCA5 produced from parent concrete suffered freeze-thaw failure and was not recommended for structural concrete as the water absorption was above 8%. The RCA could be used as structural concrete coarse aggregate when the freeze-thaw damage of NAC was smaller than 0.367.(2)The freeze-thaw damage of parent concrete also had an adverse impact on the mechanical properties and frost resistance of RAC. The frost resistance of RAC obtained from parent concrete with larger freeze-thaw damage was worse. The service life of RAC1, RAC2, RAC3, and RAC4 in cold regions can reach 70, 69, 56, and 52 years respectively, while that of RAC5 prepared from parent concrete with freeze-thaw failure was only 38 years. The weaker interfacial transition zone and porosity of RAC were the fundamental reason for the weakening of the properties of RAC.(3)The adhesive mortar of RCA played an important role in the intergenerational transfer of parent concrete freeze-thaw damage. The freeze-thaw damaged microelement dispersed in the adhesive mortar of RCA, resulting in the decline of the physical properties of RCA. When RCA was used to prepare RAC, these microelements were transferred again, which led to a decrease in the macroscopic mechanical properties and frost resistance of RAC.(4)Emergy analysis showed that the RAC production mode had more sustainable application potential than NAC. The reuse of waste concrete after freeze-thaw failure required higher economic input, higher environmental load, lower output efficiency, and sustainability. The performance, environmental load and economic benefit of RAC prepared by using waste concrete after freeze-thaw failure were inferior to that of waste concrete without freeze-thaw failure.The limitation of this study is that RAC was prepared at a 100% replacement rate, and preparations under different replacement rates were not involved. Besides, the emergy conversion rate is different under different spatial and temporal conditions, which will have an uncertain impact on the research results. With the application of emergy theory in RAC sustainable production, the emergy theory system needs more basic data to be improved. The study enriches the sustainability analysis database of recycled aggregate concrete and provides a theoretical basis for the preparation of standards. Under the background of vigorously advocating green and low-carbon economy all over the world, the sustainable application potential of modified RCA to produce RAC by green and low-carbon methods will be a hot topic in future research.


Methionine is the first limiting amino acid and plays an important role in the growth and health status of chicks. Animal health status was easily detected by examining the hematological parameters. This study aimed to investigate the influence of methionine sources (L-methionine and DL-Methionine) at different supplementation levels  on growth performance and health status of chicks. The results of the feeding trial found no differences in the chicks’ growth performance and feather growth on neither methionine sources nor methionine levels. However, most of the hematology parameters were affected by the methionine sources and/or methionine levels, as well as their interaction being significant in some cases. Therefore, this trial showed that varying methionine by ±10% of the requirements does not adversely affect growth performance and feather growth.p < 0.05). Among the treatment groups, the number of white blood cells (WBC) on 90% DL–Met was the lowest. WBC, RBC, Hb, and Ht were higher from chicks that received 100% DL-Met than all other levels, regardless of the Met sources. The MCV, MCH, and MCHC values were not affected by either Met source or levels or their interactions. Met source and level interactively affected the Ht, WBC, LYM %, and GRAN values of TSL chicks (p < 0.05). The platelet number was affected by Met source only (p < 0.05) with chicks receiving L-Met source having more than twofold higher platelet values than DL-Met source. In conclusion, varying Met levels by ±10% of the requirement does not adversely affect the growth performance, feather growth, and hematological parameters of TSL chicks reared for up to four weeks of age. DL-Met increased the body weight and improved the white blood cells, red blood cells, and hematocrit at 28 days after hatch. DL-Met showed similar biological efficacy as L-Met for body weight and feather growth but not for the hematological parameters.The study was carried out to determine whether sources or levels of methionine (Met) affect the health status of Tetra-SL (TSL) chicks by examining growth performance, feather growth, and hematological parameters. A total of ninety-six (96) day-old (44.2 ± 0.18 g lw) TSL chicks were randomly allotted to six treatment groups (three levels for each DL and L-Met source) with four replicates of four chicks each. Chicks were fed ad libitum diets supplemented with 90, 100, and 110% of methionine requirements for four weeks after hatch. The parameters examined are bodyweight (BW), average daily gain (ADG), feather length (FL), and hematological parameters, including: red blood cell (RBC) count, hemoglobin (Hb) concentration in the blood, hematocrit , number of white blood cells (WBC), platelet count, mean corpuscular volume of red blood cells (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), lymphocyte (LYM), mid-range (MID), and granulocyte (GRAN). There was no significant effect of Met sources and levels on BW, ADG, and FL of TSL chicks for the first four weeks of rearing. The RBC, Hb, Ht, WBC, LYM, MID, and GRAN values of TSL chicks were statistically influenced by dietary Met sources and Met levels ( Amino acids (aa) are one of the critical components in poultry diets, especially sulfur aa, which are essential for growth and feather development of poultry ,2. MethiIn addition, hematological parameters have been referred to for basic diagnostics of health status in mammals but have rarely been used in birds ,26. The This trial was carried out at the Institutes for Agricultural Research and Educational Farm of the University of Debrecen. The experimental protocol and procedures were reviewed and approved by the Animal Care Committee of the University of Debrecen (6/2021/DEMÁB).2/bird) per pen. The experimental design was a complete randomized block with a 2 × 3 factorial arrangement (2 Met sources × 3 Met levels). The chicks had similar initial body weight for each treatment and pen. Feed and water were provided ad libitum. The pens were bedded with wood shavings. Extra heating in the pens was provided by infrared lamps (optima plus II 175 W), and the temperature in the pens was maintained according to the breeder’s recommendation [The Tetra-SL LL (TSL) breeder eggs were obtained from Bábolna Tetra Ltd.  and incubated at standard temperature and humidity . After hatching, a total of 96 one-day-old chicks (44.2 ± 0.18 g lw) were weighed and distributed to six treatments, with four replicates having four chicks , 2 chicks from each pen  were randomly selected. Blood was collected from the cutaneous ulnar vein  into EDTA-coated tubes following the procedures of Kelly and Alworth [n = 4), while for BW, FL (n = 16), and hematological parameters (n = 8 per treatment), the individual birds were considered an experimental unit. SAS OnDemand For Academics (https://www.sas.com/it_it/software/on-demand-for-academics.html (last accessed on 21 July 2022) GLM procedure two–way analysis of variance was used to analyze all data. Tukey’s multiple comparison test was performed when analyses of variance indicated a significant difference. The p-value of p< 0.05 was considered statistically significant.Before conducting statistical analyses, data were checked for outliers by using the median absolute deviation method (MAD)  and normExcept for the interaction effect in week 4 , neitherMet source and level had no effect on feather development as a main effect; however, their interaction was significant or tended to be significant at all data collection days . Generalp = 0.004) (p = 0.016). However, there was no significant interaction between Met source and levels and the number of RBC. There was a significant difference in the Hb concentration between the Met sources (p = 0.001) and among the Met levels (p = 0.013). As with RBC, there was no significant interaction effect of Met source and levels on the Hb concentration of chicks’ blood at four-weeks of age.The number of erythrocytes (RBC) in the blood of chicks was significantly influenced by Met source (= 0.004) . The Metp = 0.005) and levels (p = 0.007) with a demonstration of the interaction of Met source and Met levels (p = 0.024). However, there were no significant differences in MCV, MCH, and MCHC values exerted by either dietary Met sources, levels, or their interaction. The dietary Met source significantly affected the platelet count in blood liters (p = 0.001) with the higher values observed in L-Met as compared to DL-Met. No significant effects of Met levels or interactions of Met sources and Met levels were revealed. The highest value of platelet count was obtained in the L-Met group with 110% inclusion (10.44 × 109/L), while the lowest was in the DL-Met group at the same dose (2.33 × 109/L)  .p = 0.003), Met levels (p = 0.009), and their interaction (p = 0.001). The percentage of lymphocytes was also significantly influenced by both dietary Met source and dietary Met levels with their interaction, the same as for WBC count. Unlike the percentage of lymphocytes, the number of LYM cells was only affected by Met sources (p = 0.044) and levels (p = 0.023) with no interaction effect observed. While the MID percentage was not influenced by the source or the levels, the MID number was significantly affected by both factors but not by their interactions (The WBC count in the blood liter was affected by both Met sources (ractions .p = 0.014) and levels (p = 0.012) with no interaction effect. However, the GRAN cell count was significantly affected by Met-source (p = 0.005) and Met levels . The highest levels of dietary inclusion in the L-Met group resulted in the lowest number and percentage  of GRAN as compared to other dietary inclusion levels in both sources  of amino acids, particularly methionine, which is the first limiting amino acid in maize- and soybean-meal-based poultry diets ,30. Our Moreover, another objective of this trial was to determine the effect of DL-Met and L-Met supplementation at different levels on the feather development (length) in TSL chicks. The results showed that neither Met source nor Met levels affected the feather length development in the chicks from the first week to the third week of rearing. However, in the fourth week, the interaction of source and level was observed. These findings are in agreement with the results reported by Chen et al.  where noThe other main objective of this study was to investigate the effects of DL-Met and L-Met on the hematological parameters of TSL chicks. Met source and levels both had a significant impact on blood parameters, both separately and in combination. No similar study was found on the effect of Met sources and levels on the TSL genotype. Hematological tests are commonly used in mammals to assess the health status of the animal but are not common in birds due to a lack of generally accepted reference values .12/L and for L-Met ranged from 2.49–3.02 × 1012/L, which are within the recommended upper limit range of 2.5–3.5 × 106/µL for healthy chickens reported by Mamba et al. [6/L, respectively. Furthermore, Tombarkiewicz et al. [6/L to 1.80–1.96 × 106/L to 2.72–2.91 × 106/L on the 1st, 7th, and 35th day of life, respectively. Higher Hb levels confirm the observed effect of Met source and levels on RBC numbers. However, the Hb values of TSL chicks amongst the six treatments ranged from 9.63 to 13.80 g/dL and are within the reference range of 7.0 to 13.0 g/dl of healthy chickens [The assessment of RBC numbers is an essential element in hematological diagnostics which detects the malfunctioning of the circulatory system . The stua et al. . Furthera et al.  and Dinga et al. , who disz et al.  found thchickens . The Ht chickens .9/L and was influenced by dietary Met sources.The elevated RBC parameters that include MCV, MCH, and MCHC are believed to be due to blood cell maturation disorders, such as megaloblastic and hemolytic anemia. However, in this study, MCV and MCH were not influenced by either Met source or Met level. This corroborates findings from the study by Chen et al.  where th3/µL [3 /µL [9/L. This result correlates with the findings of Tombarkiewicz et al. [3 /µL in response to the injection of methionine in the eggs. The influence of methionine at the 100% inclusion level had the highest WBC number than either the 90 or 110% level in both Met sources, indicating the immunological status of the chicks. The higher value of WBC suggests the ability of the chicks to fight or resist diseases, which reflects adequate methionine [Several studies have shown that the WBC count in adult chickens ranges from 12–30 × 103/µL  or 9–32 L [3 /µL , but in z et al.  in youngthionine ,46. Furtthionine  showed tthionine . GranuloIn conclusion, supplementing the pre-starter diet with 90% of the recommended Met level could maintain growth performance and support feather growth the same as 100% and 110% Met levels of either DL or L Met from 1 to 28 days of age for TSL chick. Generally, DL-Met improved the hematological parameters of TSL chicks reared to 28 days of age compared to L-Met supplementation. This research suggests that DL-Met and L-Met have similar effects on early TSL chicks’ development and feather growth, implying that they may have the same biological efficacy for average daily gain and body weight with different utilization for hematological parameter responses. The anticipated higher bioavailability of L-Met could not be justified.


IgG4-related lung disease (IgG4-RLD) is an unusual disease, with various clinical manifestations and various chest imaging findings. The patients may have no respiratory symptoms. Therefore, diagnosis is challenging. This can easily cause misdiagnosis and mistreatment.A 71-year-old male presented with chest pain, cough, and shortness of breath. Plain chest computed tomography scans showed multi-locus nodes at the center of the hilum.Percutaneous lung biopsy was performed, and IgG4-RLD was diagnosed.Prednisone was orally administered daily.The case’s symptoms improved. The patient was discharged from the hospital. After 2 months of reexamination, his symptoms were relieved. Reexamination of the chest computed tomography showed that multi-locus nodes of the lung were obviously absorbed compared with those before.IgG4-RLD is a rare respiratory disease. It has atypical clinical manifestations and chest images. We report the first case of IgG4-RLD showing multi-locus nodes centered on the hilar, hypertrophic mucosa; as well as a narrow and even occluded lumen. The most commonly involved organs are the pancreas, bile duct, main salivary gland (submandibular gland and parotid gland), lacrimal gland, retroperitoneum and lymph nodes.,4 Immunoglobulin G4 related disease was first reported in the late 1990s. Yoshida et al reported that a large number of IgG4 positive plasma cells infiltrated in a patient with autoimmune pancreas (AIP). In 2003, it was reported for the first time that a large number of IgG4-positive plasma cells infiltrated the lung lesions of patients with AIP. It has been suggested that AIP is a new autoimmune disease related to IgG4 with multiple system involvement, which may lead to lung involvement. In 2009, Zen et al called IgG4-RD involved in lung “IgG4-related lung disease (IgG4-RLD).” Owing to the late recognition of IgG4-RLD and lack of clinical experience, it is easy to cause misdiagnosis and mistreatment. Here, we report a pathologically confirmed case of IgG4-RLD with multi-locus nodes were centered on the hilum, and review the relevant literature to improve the understanding of the disease.IgG4-related disease (IgG4-RD) is a newly recognized autoimmune-mediated fibroinflammatory disease that may affect multiple organs and lead to tumefactive, tissue-destructive lesions and organ failure. It is characterized by a dense lymphoplasmacytic infiltrate rich in IgG4-positive plasma cells, storiform fibrosis, and often but not always, elevated serum IgG4 concentrations.2 and 9.2 kpa of PaO2 . No obvious abnormalities were found on an enhanced CT scan of the upper abdomen. Further electron bronchoscopy showed that the bronchial mucosa of the upper left, upper right, middle right, and lower left lobes was hypertrophic. The lumen was narrow and the apical segment of the upper right lobe was occluded  scan showed multi-locus nodes in the upper right, middle right, upper left, and lower left lobes. These shadows were centered on the hilum, with an unclear boundary. Local bronchial lumen stenosis, miliary nodular shadows in both lungs, and an enlarged hilar area were also observed . Five cases of chest imaging showed cavities, 2 cases showed cystic lesions, and 1 indicated emphysema. Imaging manifestations can occur alone or in combination with several types of lesions. The above situation shows that although the imaging manifestations of IgG4-RLD have certain characteristics, they lacks obvious specificity. Nodular shadows and mass shadows should be differentiated from lung cancer. Interstitial changes in the alveoli, enlargement of hilar/mediastinal lymph nodes, and thickening of the bronchial wall and vascular bundle should be differentiated from diffuse lung diseases such as lymphoproliferative diseases, sarcoidosis and nonspecific interstitial pneumonia.Histopathological examination is the key to the diagnosis of the disease, and tissue biopsy is important to exclude malignant diseases, tuberculosis, interstitial pneumonia, organic pneumonia, Castleman disease, and other diseases. A total of 189 patients had definite histopathology. Pathological specimens from 161 patients were obtained by pulmonary resection, video-assisted thoracic surgery, lung biopsy, percutaneous lung biopsy, and bronchoscopy lung/mucosa biopsy. Two cases were diagnosed by pleural biopsy, and 19 by extrapulmonary organ biopsy. Although there were no lung tissue biopsy data, the lung images decreased or even disappeared following treatment; thus, IgG4-RLD was confirmed. Histopathological features: in 184 biopsies, lymphocytic plasmacytosis was observed in 132 biopsies, IgG4 positive plasmacytosis was found in 10 to 159 (number/ HP). The IgG4/IgG plasma cell ratio in 156 tissues was between 23% and 88%. There were obliterative phlebitis in 49 tissues, obliterative arteritis in 23 tissues, storiformfibrosis in 82 tissues and irregular fibrosis in 38 tissues. In addition, a small number of IgG4 positive cells can also be seen in many inflammatory infiltrates; therefore, in absolute quantity, the number of IgG4 positive plasma cells determined in the literature is between 10 and 50/high power field. If the ratio of IgG4 positive plasma cells to IgG plasma cells is >50%, it would be more helpful in diagnosing IgG4-RD. In particular, in the late stage of the disease, when only a small number of plasma cells exist in the tissue and fibrosis becomes the main component, the ratio of IgG4 to IgG plays a key role in the diagnosis.IgG4-RLD responded well to glucocorticoid therapy. More than 50% of the 189 patients reviewed in this study were treated with glucocorticoids alone, with an effective rate of 87.6%. Commonly used glucocorticoids include prednisone, prednisolone or methylprednisolone, with an initial dosage of 0.5 to 1 mg/kg/day for prednisone, 0.5 to 0.6 mg/kg/day for prednisolone or 0.4 to 80 mg/kg/day for methylprednisolone. A total of 9.3% of patients experienced recurrenced after glucocorticoid reduction, and the symptoms improved after glucocorticoid addition or combination therapy with immunosuppressive agents. The immunosuppressive agents used in this review included methotrexate, mycophenolate mofetil, azathioprine, cyclophosphamide, and cyclosporine. The effective surgical rate was 56%. Fourteen cases were untreated, and 2 cases were self-healing , indicating that the disease had some self-healing. In particular, fluticasone propionate (500 μg) inhaled with salmeterol (50 μg) was administered twice a day. Two cases of glucocorticoids combined with rituximab have been reported in the literature. Although they were effective, the disease relapsed easily. When the drugs were reduced, the disease recurred easily. Currently, the optimal maintenance dose and time for IgG4-RLD have not been determined. After treatment, patients who stop the drug and relapse can be treated with glucocorticoids again. Immunosuppressive agents should be used to maintain remission. However, the prognosis of this disease remains unclear. Of the 189 patients reviewed in this study, only 3 died and 1 died after video-assisted thoracic surgery.In summary, IgG4-RLD is a rare respiratory disorder. It has atypical clinical manifestations and chest images. It can exist alone and is often associated with other systemic diseases. Moreover, it is difficult to distinguish it from common respiratory diseases, such as lung cancer, tuberculosis, and the diagnosis of the disease depends on clinical, chest images, serum IgG4 levels, and comprehensive pathological analysis. The patient responded well to glucocorticoid treatment. Some cases are prone to recurrence in the process of hormone reduction; therefore, they should be followed up for a long time after treatment. However, its prognosis is unclear. Therefore, further prospective multicenter clinical studies are required.Our case implied 3 clinical evidences: first, multi-locus nodes 1n the middle of the hilar could be the presentation of IgG4-related respiratory disease. The second is, IgG4-related respiratory disease with hypertrophic mucosa. Finally, IgG4-related lung disease can be very sensitive to prednisone. Lung lesions were absorbed further, the serum concentration of IgG4 decreased after prednisone treatment, and the symptoms significantly improved in our case. In brief, we report the first case of IgG4-RLD showing multi-locus nodes centered on the hilar, hypertrophic mucosa, as well as a narrow and even occluded lumen. These findings further our understanding of the characteristics of IgG4-RLD.HR contributed to the conception and design, all data collection, and analysis and interpretation of data and played a major role in the writing of the article. ZH, JW and SM provided raw data of the patient. XZ provided suggestions for documenting the pathological findings in this report, and CZ made key revisions to the manuscript for important intellectual content. All authors have read and provided consent for the final manuscript, and have ensured the integrity of their respective intellectual parts.Conceptualization: Hongmei Ren.Data curation: Hongmei Ren.Formal analysis: Hongmei Ren.Funding acquisition: Hongmei Ren.Investigation: Hongmei Ren, Jinping Wang, Siming Meng.Methodology: Hongmei Ren.Project administration: Hongmei Ren.Resources: Hongmei Ren, Zhiqing Han, Xiaoming Zuo, Jinping Wang, Siming Meng.Supervision: Hongmei Ren, Cuixia Zheng.Validation: Hongmei Ren.Writing – original draft: Hongmei Ren.Writing – review & editing: Hongmei Ren, Cuixia Zheng.