Tachypleus tridentatus) has been considered as food and traditional medicine for many years. Kynurenic acid (KA) was isolated from horseshoe crab in this study for the first time in the world. A previous study in 2018 reported that intraperitoneal administration of KA prevented high-fat diet- (HFD-) induced body weight gain. Now, we investigated the effects of intragastric gavage of KA on HFD mice and found that KA (5 mg/kg/day) inhibited both the body weight gain and the increase of average daily energy intake. KA reduced serum triglyceride and increased serum high-density lipoprotein cholesterol. KA inhibited HFD-induced the increases of serum low-density lipoprotein cholesterol, coronary artery risk index, and atherosclerosis index. KA also suppressed HFD-induced the increase of the ratio of Firmicutes to Bacteroidetes . KA partially reversed HFD-induced the changes in the composition of gut microbial genera. These overall effects of KA on HFD mice were similar to that of simvastatin (positive control). But the effects of 1.25 mg/kg/day KA on HFD-caused hyperlipidemia were similar to the effects of 5 mg/kg/day simvastatin. The pattern of relative abundance in 40 key genera of gut microbiota from KA group was closer to that from the normal group than that from the simvastatin group. In addition, our in vitro results showed the potential antioxidant activity of KA, which suggests that the improvement effects of KA on HFD mice may be partially associated with antioxidant activity of KA. Our findings demonstrate the potential role of KA as a functional food ingredient for the treatment of obesity and hyperlipidemia as well as the modulation of gut microbiota.The marine horseshoe crab ( Tachypleus tridentatus) has been known as “living fossils” [Tachypleus Amebocyte Lysate (TAL), tachyplesin, and the technology of extracting chitin from horseshoe crab shells. However, there are few reports on the composition and chemical properties of horseshoe crab. To the best of our knowledge, kynurenic acid (KA) was isolated from horseshoe crab tail for the first time in this study.Horseshoe crab  in the human intestine. Gut microbiota has a key regulatory role for the body's metabolism and is highly related with human diseases, including atherosclerosis [Compared with other areas in human, gut microbiota has both the largest numbers of bacteria and the highest number of species types. Gut microbiota is mainly composed of intestinal flora, and there are about 10clerosis –27. Gut clerosis . With moclerosis . The oppBased on the two previous studies about KA, we speculated that oral KA might have the beneficial effects on high-fat diet (HFD) model. First, a previous study in mice in 2018 reported that the intraperitoneal (i.p.) administration of KA (5 mg/kg/day) prevents body weight gain induced by HFD . Feed ofIn this study, the compound of KA was isolated and purified from horseshoe crab tail by modern spectral analysis methods, such as TLC, UV, NMR, and ESI-MS. The antioxidant activities of KA from horseshoe crab were determined. The effects of KA on food intake, energy intake, body weight, and serum lipids were investigated in the HFD mouse model. This study also explored the possible effects of KA on gut microbiota in HFD mice. We selected simvastatin (SV) as a positive control .Tachypleus tridentatus) was collected from the North Gulf, Xiamen, China. Reversed phase silica gel YMC-ODS C18 was purchased from YMC . Silica gel chromatography plates GF254 were obtained from Qingdao Ocean Chemical Plant . We prepared normal phase column chromatography silica gel (Silia Flash P60) from SiliCycle , Sephadex LH-20 from GE Healthcare , and deuterated solvents for NMR experiments from Shanghai Macklin Biochemical Co. Ltd. . Methanoic acid and methanol for ESI-MS were purchased from TEDIA Biochemical Co. Ltd. (USA). For FRAP and ABTS assay, we used total antioxidant capacity assay kit from Beyotime Institute of Biotechnology . We acquired all other reagents with analytical grade from Sinopharm Chemical Reagent Co. .Horseshoe crab (w/v) at 40°C for 24 h. The extracts were combined, concentrated, and evaporated for dryness by a rotary evaporator  at 45°C. The methanol extract of horseshoe crab tail was dissolved in a little amout of methanol and then filtered using a Dismic-25 JP membrane filter with pore diameter of 0.45 μm . Finally, the filtrate was evaporated to concentrate the crude extract.The dried horseshoe crab tail was crushed and further extracted by three times with methanol in a ratio of 1 : 10 (The methanol extract was separated into 4 fractions by silica gel column chromatography (3.6 × 25 cm) with gradient elution of water and methanol from (1 : 0 to 0 : 1). The fraction 2 was applied to Sephadex LH-20 column chromatography (2.5 × 150 cm) with methanol. The fraction was subjected to TLC analysis and then to reverse silica gel column chromatography by gradient elution of water and acetone from 4 : 6 to 0 : 1, which was separated into fraction A and fraction B. The fraction B was repeatedly recrystallized three times to finally obtain the compound.1H-NMR, 13C-NMR, 1H-1H COSY, HMBC, and HSQC (400 MHz) spectra of the compound by a Bruker AVANCE-400 NMR spectrometer . The mass spectra was analyzed by ESI-MS with Applied Biosystems .The purified component HCT8 was dissolved in a trace amount of methanol and placed in a clean nuclear magnetic tube. The methanol was subjected to rotary evaporation with a rotary evaporator; the dry weight was weighed and labeled and then dissolved with the corresponding deuterated reagent. We recorded μL) were mixed with fresh ABTS working solution (200 μL) at room temperature. After 6 min, the absorbance (at 734 nm) from the mixtures was recorded. We used Trolox as a reference compound. On the other hand, the total antioxidant capacity of KA was evaluated using the assay kit for ferric reducing ability of plasma (FRAP) [μL) were mixed with working solution of FRAP (180 μL). After 5 min, the absorbance (at 593 nm) of the mixtures was recorded. We prepared the standard curve with FeSO4. Finally, we showed the values expressed as the mean of 3 measurements.With 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) assay kit , 33, we ance at 7 nm from a (FRAP) . We prepKunming (KM) mice weighing 18–20 g  were obtained from Beijing HFK Bioscience Co. Inc . They were placed under standard laboratory conditions . This study was approved through the Institutional Animal Care and Use Committee from Animal Laboratory Center of Jimei University (SCXK 2016-0006). All experiments met the requirements of the National Laboratory Animal Act (China). According to the principles of the 3Rs , for decreasing the number of mice used and their suffering, every possible effort into experimental animal design from this study has been adopted. After random grouping, the first group of mice (NC) received the normal chow diet with calorie of 3.42 kcal/g, and the other four groups received HFD  . The firThe serum could be obtained by centrifugation from blood for 20 min at 10000 g and at 4°C. TC, TG, LDL-C, and HDL-C levels from serum were determinated with commercially available kits  and analyzed by fully automatic bioanalysis machine  . CoronarWith a modified method from the previous study , we extrP < 0.05 showed a statistically significant difference. P < 0.01 and P < 0.001 further showed highly significant difference.Data in this study were represented as mean ± standard deviation (SD) and further analyzed with Graphpad Prism 6 . The significance was determined by ANOVA followed by Duncan's multiple-comparison tests. 2+. Based on the 10 carbon signals from the 13C-NMR  spectrum  showed 5 proton signals at δ7.41 , δ7.72 , and δ7.83 , which were conjugated to each other. Proton δ 8.24 was the -OH proton signal. The ESI-MS and NMR data from KA  from 18 fecal samples after 8 weeks for understanding the effect of the KA on the composition in gut microbiota of HFD mice. We showed effective tags from sequencing data in  Figures  were obs Figures . Taxon-b Figures . Compare Figures . Compare Figures . CompareTachypleus tridentatus) tail, and it was determined to be KA , hyperlipidemia , P < 0.01), P < 0.01), and P < 0.05)), and changes of gut microbiota  and P < 0.05 to P < 0.001 for 6 microbial genera, respectively)) in mice. In this study, there were statistically significant differences for the above three phenomena between KA and HFD groups, which supported the finding that the beneficial effects of oral supplementation of KA on HFD mice. But certainly, the detailed cellular mechanisms of oral KA on HFD model still need to be further explored in future study. The i.g. of 5 mg/kg/day KA (KAH) for 8 weeks did not cause any obvious side effects in general appearance of HFD mice, which is consistent with two previous reports [Compared with the NC group, we found that HFD caused the significant obesity ( reports , 59. Die reports . Importa reports , 60. The reports . AnotherOur findings demonstrate KA has a potential functional food ingredient role for treating obesity and hyperlipidemia as well as the modulation of gut microbiota. This study, involving identification of KA as a bioactive component from the horseshoe crab, also helps to find substitutes for these creatures, which will promote the conservation of these creatures.


High prevalence of sleep problems have been reported in children with Autism Spectrum Disorder (ASD). This study aims to investigate the sleep conditions of ASD children in China, and explore the relationship between the common sleep problems and core symptoms and developmental levels.Using a cross-sectional design, we included 2 to 7-year-old children from 13 cities in China: 1310 with ASD and 1158 with typically-developing (TD) children. The neurodevelopmental level was evaluated with the revised Children Neuropsychological and Behavior Scale (CNBS-R2016). ASD were diagnosed with DSM-5 and Child Autism Rating Scale (CARS). the Social Responsiveness Scale (SRS), the Autism Behavior Checklist (ABC) and the communication warning behavior sub-scale in CNBS-R2016 valued autism behaviors. The children’ s sleep habits questionnaire (CSHQ) assessed sleep conditions.p < 0.01), and among them the four dimensions with the highest prevalence of sleep problems were bedtime resistance (25.6%), sleep anxiety (22.7%), sleep onset delay (17.9%) and daytime sleepiness (14.7%). ASD children with sleep onset delay or sleep anxiety had higher ABC, SRS scores and higher scores on communication warning behavior with sleep anxiety, with daytime sleepiness had higher ABC, SRS and CARS scores, and with bedtime resistance had higher SRS total scores. Differences in the neurodevelopmental level were not significant.The prevalence of sleep disorders in ASD children was significantly higher than that in TD .The study was approved by the ethics committee of the Children’s Hospital of Chongqing Medical University, Approval Number: (2018) IRB (STUDY) NO. 121, and registered in the Chinese Clinical Trial Registry (Registration number: The online version contains supplementary material available at 10.1186/s12888-021-03405-w. Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder that seriously endangers human health. Deficits in social communication and repetitive and stereotyped interests and behaviors are its characteristics . The latThese sleep problems can cause a series of effects. Sleep disorders may adversely affect children’s daily function, affecting behavior, learning, memory regulation and cognition –20. It mBehavioral insomnia in children is considered the most common cause of sleep disorders in children. Also in ASD. So far, behavioral interventions, sleep education, environmental changes and exogenous melatonin are the most effective ways to promote sleep in ASD . The AutThis research is part of the China Multi-Center Preschool Autism Project (CMPAP) , and colThe sample size calculation used the formula of two independent sample rates:It was reported that the highest prevalence of sleep problems in the ASD was 80%, and in TD was 50%. Set alpha was 0.05, beta was 0.1, and missing rate was 10%. The sample size required for each groups was 57 in each centers through calculation.ChiCTR2000031194).All participants’ parents signed an informed consent forms. The study was approved by the ethics committee of the Children’s Hospital of Chongqing Medical University, Approval Number: (2018) IRB (STUDY) NO. 121, and registered in the Chinese Clinical Trial Registry , residence , parental education level , annual family income .The Children’s sleep habits questionnaire (CSHQ) is an international standard questionnaire for the assessment of children’s sleep. CSHQ consisted of 33 scoring items and divided into eight dimensions: bedtime resistance, sleep onset delay, sleep duration, sleep anxiety, night waking, parasomnia, sleep disordered breathing and daytime sleepiness. Parents were asked to recall sleep behaviors occurring in a recent week  . CSHQ haDSM-5 is a clinical standard for the diagnosis of ASD, and the diagnosis must meet the following standards: Persistent deficits in social communication and social interaction (Criterion A), and restricted, repetitive patterns of behavior, interests, or activities (Criterion B) currently or by history; These symptoms are present from early childhood and limit or impair everyday functioning (Criteria C and D); These disturbances are not better explained by intellectual disability (Criteria E); according to the intensity of support required, the severity is divided into 3 levels .CARS is a scale used by professionals to assess the symptoms and severity of autism. There are 15 items in total, and each item is rated as 1 point , 2 points , 3 points , and 4 points , respectively. A score between 30 and 36 indicates mild-moderate autism and a score above 36 indicates severe autism . The useSRS is a scale for parents to fill in the daily social situation of children, concluding 65 items and consisting of 5 sub-scales  . The scoscore for typically-developing children should be < 65. The Chinese version of SRS shows that Cronbach’s alpha coefficient is 0.871 ~ 0.922, retest reliability is 0.81 ~ 0.94, and the convergent validity is 0.302 ~ 0.647 .ABC is completed by parents to assess autism symptoms. There are 57 items in total, including 5 factors such as sensory stimuli, sensorial relating, body and object use, language and social self-help. The score for each item is between 1 and 4. Scores for children without autism should be < 53 . ABC scaCNBS-R2016 can evaluate the neurodevelopmental levels and autism symptoms of children, including six domains . The developmental quotient less than 70 indicates a developmental delay and communication warning behavior quotient more than 30 is suspected of being at risk of autism . The CNBp < 0.05.SPSS statistical software 25.0 was used for statistical analysis. Shapiro-Wilk test, histogram, and QQ graph were combined to detect data normality. Continuous variables were described as mean ± standard deviation (M ± SD), medians (inter-quartile ranges) (M (IQR)) and medians (5th percentile to 95th percentile) (P5-P95). Categorical variables were described as frequencies and percentages. Two-sample independent T-test,the chi-square test and Mann-Whitney test were used for comparisons between groups. Logistic regression was used to examine the differences in sleep problems between ASD and TD groups. Significance was presumed at P < 0.05). Differences were also significant in region, residence, parental education level and annual family income . Logistic regression can be used to adjust for confounding factors. Therefore, we used it to compare the differences between the two groups. In Model I (unadjusted), the occurrence of sleep disorders was taken as the dependent variable and grouping (ASD and TD) as the independent variable . Model II included adjusted factors such as gender, age, region, residence, parents’ educational level and annual family income as independent variables, and the results showed that the differences in sleep problems between the two groups remained   children. According to the inclusion and exclusion criteria of our study. A total of 2726 children aged 2–7 years were enrolled in the study, and 2468 children were included eventually after excluding 258 children who had a sleep questionnaire loss , and the total score of CSHQ was also higher (43(36–58) VS. 41(36–51), P < 0.001). The prevalence and scores of eight dimensions of sleep problems in ASD children was higher than that in TD: bedtime resistance  VS. 9(7–13), P < 0.001), sleep onset delay  VS. 1(1–3), P < 0.001), sleep duration  VS. 3(3–6), P < 0.001), sleep anxiety  VS. 6(5–8), P < 0.01), night waking  VS. 3(3–5), P < 0.001), parasomnias  VS. 8(7–10), P > 0.05), sleep disordered breathing  VS. 3(3–4), P < 0.001), daytime sleepiness  VS. 11(8–17), P < 0.001).As shown in Table Among children with ASD, the four dimensions with the highest prevalence of sleep problems were bedtime resistance (25.6%), sleep anxiety (22.7%), sleep onset delay (17.9%) and daytime sleepiness (14.7%), respectively. The same was true for TD, with prevalence of 15.9, 15.7, 12.8 and 6%, respectively. This was consistent with previous research. However, the prevalence of night waking in ASD is the lowest in our study. Most previous reports have shown that night waking is also a significant sleep problem in ASD.P < 0.05), but lower scores in the ABC language sub-scale (P < 0.05) and CARS (P < 0.01). ASD children with sleep anxiety or sleep onset delay had higher ABC scores (P < 0.01) and SRS scores (P < 0.001), and ASD children with sleep anxiety had higher communication warning behavior scores (p < 0.05); there were no significant differences in CARS scores. ASD children with daytime sleepiness had higher ABC scores (P < 0.001), SRS scores (P < 0.001), CARS scores (P < 0.05). , and the scores and prevalence of sleep problems in all dimensions were also higher. These emphasized the severity of sleep problems in ASD. Further analysis found that the first four were bedtime resistance (25.6%), anxiety (22.7%), sleep onset delay (17.9%) and daytime sleepiness (14.7%). Except for these four dimensions, in previous reports, night waking is also a common sleep problem reported by parents, but it was the lowest in our study (5%). This sleep pattern is similar to a recent survey in China and India. China investigated the sleep conditions of 475 preschool children with ASD and found that the prevalence of sleep problems was 81.7%. The four with the highest were sleep resistance (90.9%), sleep anxiety (91.7%), daytime sleepiness (60.7%) and sleep onset delay (59.1%), and the lowest two were night waking (25.4%) and Sleep disordered breathing (19.8%) . In IndiThere are currently a large number of studies on the overall sleep status and autism symptoms of ASD, so we do not separately analyze and discuss it, but focus the research on different dimensions. For this study, it is the four dimensions of with the highest prevalence –46.The sleep problem with the highest prevalence of ASD in this survey was bedtime resistance (25.6%). Children with it had higher SRS scores, which were mainly manifested in social awareness, cognition and communication. Hollway  analyzedIn this study, ASD children with sleep onset delay and sleep anxiety had higher ABC, SRS and autism warning behavior scores, which were specifically manifested in higher social interaction, communication, cognition, language and stereotyped behavior scores. Tudor  found thThe daytime state is closely related to the study and function during the day. Our research found that daytime sleepiness was related to the core symptoms of autism and children showed higher scores in ABC, SRS, and CARS. A recent study in India found that the severity of autism was positively correlated with daytime sleepiness in male children between 2 and 6 years old . AnotherIn addition, Gender-stratified analysis showed that ASD children with common sleep problems exhibited higher ASD symptom scores both in male and female, but some results were not statistically significant in female. This may be caused by the small sample size of the girls group. The analysis found that the sample size of boys was 1077 and that of girls was 233.These four types of sleep problems were found to be related to core symptoms, but not related to the neurodevelopmental level. Previous studies on the relationship between developmental level and core symptoms are inconsistent. Several studies reported that the neurodevelopmental level or intelligence may affect sleep , 27, 29.In addition to helping to understand the sleep conditions of Chinese children with ASD, our research also provides directions for precise intervention in the future. Bedtime resistance was a sleep problem closely related to sleep behavior and sleep hygiene, and the most prominent sleep problem in this survey. Daytime sleepiness and sleep onset delay were likely to be related to abnormal melatonin levels. This suggests that in the future intervention, the cultivation of sleep hygiene and behavior is as important as the intervention of biological factors, especially starting from the actual situation of our country, combining our country’s culture and resources to formulate intervention strategies. Moreover, there is still a clinical phenomenon that children with ASD often come to the doctor because of language delay. Parents generally pay more attention to the core symptoms of the child, thus ignoring the co-occurring sleep problems which are related to core symptoms. Therefore, we suggest that in the future diagnosis and treatment, sleep assessment should be routinely included to achieve early prevention, detection and intervention of sleep disorders in ASD children.This study has limitations. First, Children are recruited online rather than randomly sampled, which may make the sample less representative, and it was designed as a cross-sectional study when comes to the relationship between sleep disorders and core symptoms, which could not establish a causal relationship. Moreover, there was no objective measure of sleep. The CSHQ was completed by parents. This could led to a response bias. So there may be deviations between the real sleep situation and the survey results. It is better to combine subjective and objective assessments when possible. Although the scale assessment was completed by trained professionals, this study involved 13 cities in China, and the quality control was not mature enough, which may also be the reason for the contrary results in the analysis of sleep resistance. Therefore, further objective research is needed.The prevalence of sleep problems in ASD is significantly higher than that in typically-developing children at all levels in China. The first four are bedtime resistance, anxiety, sleep onset delay and daytime sleepiness. These sleep problems may be not related to the developmental level with ASD, but be related to their core symptoms. In the clinic, sleep assessment should be included in the routine of ASD visits, and during the intervention, sleep hygiene education is as important as the treatment of biological factors.Additional file 1: Table S1. Difference in the prevalence of sleep disorder between ASD and TD children after adjusting confounders by logistic regression.Additional file 2: Table S2. Comparison of demographic characteristics between lost samples and included samples.Additional file 3: Table S3. Differences in autism syptoms in ASD boys* with and without common sleep problems.Additional file 4: Table S4. Differences in autism syptoms in ASD girls* with and without common sleep problems.Additional file 5: Table S5. Differences in developmental quotient in ASD boys with and without common sleep problems.Additional file 6: Table S6. Differences in developmental quotient in ASD girls with and without common sleep problems.


Background: Radioiodine-refractory differentiated thyroid cancer (RR-DTC) has a low 10-year patient-survival rate and is challenging to treat. Lenvatinib is a multikinase inhibitor approved for the treatment of RR-DTC. This study aims to assess Eastern Cooperative Oncology Group performance status (ECOG PS) and neutrophil-to-lymphocyte ratio (NLR) as prognostic markers for patients with RR-DTC treated with lenvatinib.Methods: In this retrospective analysis of the Study of (E7080) LEnvatinib in Differentiated Cancer of the Thyroid (SELECT), patients randomly assigned to receive lenvatinib were classified according to baseline ECOG PS (0 or 1) or baseline NLR (≤3 or >3). The effects of baseline ECOG PS and NLR on progression-free survival (PFS), overall survival (OS), and objective response rate (ORR) were evaluated. In addition, the effects of baseline ECOG PS on the change in diameter of target lesions and correlations between baseline NLR and the sums of the diameters of target lesions were calculated.Results: Among patients who received lenvatinib, patients with a baseline ECOG PS of 0 had statistically improved PFS , OS , and ORR  compared with patients with a baseline ECOG PS of 1. Patients who received lenvatinib with a baseline NLR ≤3 also had improved PFS  and OS  versus patients with a baseline NLR >3. Moreover, patients with a baseline NLR ≤3 had a trend toward increased ORR  compared with patients with a baseline NLR >3. Treatment-emergent adverse events were generally similar among patients who received lenvatinib, irrespective of patients' ECOG PS at baseline.Conclusion: Lower ECOG PS and NLR may provide prognostic value for improved efficacy in patients with RR-DTC. ClinicalTrials.gov no. NCT01321554. The incidence of cancer with thyroid as the site of origin has increased from 3.6 cases per 100,000 in 1973 to 8.7 cases per 100,000 in 2002, representing a 2.4-fold increase in the number of cases diagnosed/year . AlthougStudy of (E7080) LEnvatinib in Differentiated Cancer of the Thyroid (SELECT)  ,13. In S(SELECT) .Despite this advancement in treatment, few prognostic factors have been described for RR-DTC. Eastern Cooperative Oncology Group performance status (ECOG PS)—a comprehensive measure of a patient's level of functioning —is assocpost hoc analysis assessed baseline ECOG PS and NLR as potential prognostic indicators in patients with RR-DTC treated with lenvatinib.Treatment with tyrosine kinase inhibitors in RR-DTC is not curative and is associated with treatment-emergent adverse events (TEAEs); therefore, identification of prognostic markers could help physicians make the challenging decision of whether to initiate systemic therapy. This exploratory SELECT was a phase 3 randomized, double-blind, multicenter study that compared lenvatinib versus placebo in patients with RR-DTC. Full details of the study have been published . A briefAll patients provided written informed consent, and the study protocol was approved by all relevant institutional review bodies. The study was conducted in accordance with the provisions of the Declaration of Helsinki and local laws.In SELECT, patients were randomly assigned (2:1) to receive oral lenvatinib at 24 mg/day or placebo in 28-day cycles . Tumor rpost hoc subgroup analysis included patients from SELECT who were randomly assigned to receive lenvatinib, using the data cutoff date from the primary analysis . Patients were then divided into subgroups according to baseline ECOG PS (0 or 1) and baseline NLR of ≤3 or >3 . NLR was calculated based on peripheral blood cell counts. As corticosteroid treatment can impact NLR values, a separate analysis was conducted excluding 20 patients who were receiving concomitant corticosteroids at baseline to confirm the accuracy of the NLR data.This exploratory Efficacy analyses were conducted with patients who received lenvatinib and were grouped by either ECOG PS or NLR. The effects of baseline ECOG PS and baseline NLR on PFS, OS, and objective response rate (ORR) were evaluated separately. In addition, the effects of baseline ECOG PS on the change in the sums of the diameters of target lesions and correlations between baseline NLR and the sums of the diameters of target lesions were calculated. The number of metastatic sites involved was assessed in patients with a baseline ECOG PS 0 versus 1, as well as NLR ≤3 versus NLR >3. ECOG PS was also measured in patients with a baseline NLR ≤3 and NLR >3. Kaplan–Meier analyses for OS and PFS were also conducted on data from patients who had received placebo, grouped by either ECOG PS or NLR. These results are reported in the p-values were based on an unstratified log-rank test. The CIs for ORR were calculated by using asymptotic normal approximation; the odds ratios (OR) between subgroups and p-values were calculated by using the Chi-square test. Percentage changes from baseline to postbaseline nadir in the sums of diameters of target lesions and percentage changes over time according to ECOG PS at baseline (0 or 1) were plotted and assessed.Statistical analyses were performed using SAS (SAS Institute Inc.) version 9.4. PFS and OS were summarized by using Kaplan–Meier estimates; 95% confidence intervals (CIs) of the median were constructed with a generalized Brookmeyer and Crowley method; hazard ratios (HRs) between subgroups were estimated from an unstratified Cox proportional hazard model; and et al  is described as per Hinkle et al .post hoc nature of the analyses in this article, all reported p-values  should be considered nominal.Given the The baseline demographic and disease characteristics of patients from SELECT who were randomly assigned to receive lenvatinib were analyzed according to patients' ECOG PS and are shown in p = 0.2414). The baseline numbers of metastatic sites analyzed according to NLR for patients randomly assigned to receive lenvatinib are shown in p = 0.0011). In general, no significant correlations were observed between age and NLR or age and ECOG PS.Patients in the lenvatinib arm were also analyzed according to NLR. A baseline NLR ≤3 was recorded for 121 (46.4%) patients, and 140 (53.6%) patients had a baseline NLR >3; the median baseline NLR for patients randomly assigned to receive lenvatinib was 3.1 (range: 0.7–36.3). Absolute neutrophil and lymphocyte counts by NLR are reported in p = 0.001) (p = 0.0004)  . Similar 0.0004) . Tumor rp < 0.0001). Percent changes in the sums of diameters of target lesions from baseline to postbaseline nadir according to baseline ECOG PS are shown in The ORR in patients with an ECOG PS of 0 was 78.5% [CI 71.8–85.2] versus 51.0% [CI 41.4–60.6] in patients with an ECOG PS of 1  (p = 0.0029)  were excluded from analysis, median PFS and OS values by baseline NLR were similar to those of the overall lenvatinib patient population  . Patient 0.0029) . When papulation .p = 0.08). Further, among patients without concomitant corticosteroid use at baseline, patients with an NLR ≤3 (n = 116) had an ORR of 71.6% [CI 63.3–79.8], and patients with an NLR >3 (n = 125) had an ORR of 63.2% [CI 54.8–71.7]   . Overall = 0.17) .p = 0.0032) and OS (p = 0.0012) (p = 0.0587), but was not significantly associated with OS (p = 0.4145). Baseline NLR was significantly associated with PFS (p = 0.0009) and OS (p = 0.0128). Patient age at baseline was significantly associated with PFS (p = 0.0459), but not with OS (p = 0.2125).According to multivariate analyses, baseline ECOG PS (0 vs. 1) had a significant association with PFS ( 0.0012) . The sumPFS and OS data by baseline ECOG PS for patients who received placebo are provided in The median duration of treatment with lenvatinib was 14.3 months (range: 0.8–26.8) for patients with a baseline ECOG PS of 0 and 10.6 months (range: 0.2–26.5) for patients with a baseline ECOG PS of 1. The median dose intensity of lenvatinib was 16.2 mg/day/patient (range: 5.8–24.0) for patients with an ECOG PS of 0 at baseline and 17.6 mg/day/patient (range: 6.5–24.0) for patients with an ECOG PS of 1 at baseline.An overview of TEAEs and the most common TEAEs by baseline ECOG PS are summarized in post hoc analysis of SELECT provides insights into markers of efficacy among patients with RR-DTC treated with lenvatinib. Specifically, patients with lower ECOG PS and lower NLR   . In an e< 0.001) . Our exppost hoc nature of the assessment. In addition, concurrent corticosteroid treatment can increase NLR  were also improved in patients with lower ECOG PS. A further limitation is that this study was not powered to assess the predictive validity of ECOG PS or NLR; therefore, additional studies are needed to determine whether any of these indicators are predictive for lenvatinib versus placebo. Finally, ECOG PS may be assessed inconsistently by clinicians and may be unreliable as a prognostic marker, particularly in a real-world setting .Despite these limitations, this analysis indicates that correlates of efficacy, including ECOG PS and NLR, may provide prognostic value and treatment guidance in patients with RR-DTC. For clinicians, deciding on the optimal time to initiate tyrosine kinase inhibitor therapy can be challenging because of the desire to avoid AEs , but thi


Tuberculosis (TB) has been a major public health problem in South Korea. Although TB notification rate in Korea is gradually decreasing, still highest among the member countries of the Organization for Economic Cooperation and Development. To effectively control TB, understanding the TB epidemiology such as prevalence of latent tuberculosis infection (LTBI) and annual risk of TB infection (ARI) are important. This study aimed to identify the prevalence of LTBI and ARI among South Korean health care workers (HCWs) based on their interferon-gamma release assays (IGRA).This was single center, cross-sectional retrospective study in a tertiary hospital in South Korea. We performed IGRA in HCWs between May 2017 and March 2018. We estimated ARI based on IGRA results. Logistic regression model was used to identify factors affecting IGRA positivity.A total of 3233 HCWs were analyzed. Median age of participants was 38.0 and female was predominant (72.6%). Overall positive rate of IGRA was 24.1% and IGRA positive rates age-group wise were 6.6%, 14.4%, 34.3%, and around 50% in the age groups 20s, 30s, 40s, and 50s and 60s, respectively. The ARIs was 0.26–1.35% between 1986 and 2005; rate of TB infection has gradually decreased in the last two decades. Multivariable analysis indicated that older age, healed TB lesion in x-ray, and male gender were risk factors for IGRA positivity, whereas working in high-risk TB departments was not.Results showed that ARI in South Korean HCWs gradually decreased over two decades, although LTBI remained prevalent. Our results suggest that the LTBI test result of HCWs might be greatly affected by age, rather than occupational exposure, in intermediate TB burden countries. Thus, careful interpretation considering the age structure is required.The online version contains supplementary material available at 10.1186/s12879-021-06855-5. South Korea is an intermediate tuberculosis (TB) burden country; in 2019, the annual incidence of TB was reported to be 59 per 100,000 ; howeverTo date, LTBI prevalence studies and analysis of ARI have been conducted mainly using the tuberculin skin test (TST) in South Korea , 6, 7. IIn this study, we aimed to identify the prevalence of LTBI and estimate ARI in health care settings based on IGRA results of HCWs. We also aimed at identifying the risk factors of LTBI for HCWs with a particular focus on their age in an intermediate TB burden country.This study was conducted at Severance Hospital, a 2500-bed tertiary referral hospital in South Korea. A total of 3283 HCWs underwent the LTBI test between May 2017 and March 2018 as part of a national TB elimination program. The LTBI test used was QuantiFERON® TB-Gold In Tube test (QFT-GIT). High-risk departments such as respiratory department and outpatient clinic, medical intensive care unit, emergency department, microbiology laboratory, and radiology department were defined as TB-related departments. We collected data related to participants’ age, sex, occupation, hospital department, and years of employment; other electronic medical records were retrospectively reviewed. Previously healed TB lesions on chest radiographs were defined as calcified nodules or lymph nodes, discrete fibrotic scars with volume loss, or pleural thickening or calcification The IGRA was performed using a QuantiFERON® TB-Gold In Tube test  following the manufacturer’s instructions. A positive IGRA result was defined as an interferon-gamma response to the TB antigen minus that of the nil tube of ≥ 0.35 IU/mL. Chest radiographs were evaluated to exclude active TB at the same period. The HCWs were diagnosed with LTBI if the IGRA results were positive.[1/mean age of each age group]) × 100ARI (%) = (1 − [1 − IGRA positive rate of each age group/100]Midyear of TB infection (y) = 2017-mean age of each age group/2The study subjects were divided into nine age groups . ARIs were calculated based on the results of IGRAs using the following math formula ARI (%) =t-test or the Mann–Whitney test. Categorical variables were reported as number and percentage, and were analyzed using the chi-square test or Fisher’s exact test. To determine if IGRA positivity differed according to age in this study, we obtained the probability of a positive result in each subject using logistic regression and expressed it in the form of a figure. Risk factors associated with IGRA positivity were investigated using logistic regression models, and the results were reported as odds ratios (ORs) and 95% confidence intervals (CIs). All tests of significance were 2-tailed, and a p value of less than 0.05 was considered statistically significant. All analyses were performed using SPSS software, version 23.0  and SAS 9.4 .Continuous variables were presented as means ± standard deviation (SD), or median and interquartile range (IQR), and were analyzed using the Student Overall, 3233 HCWs were analyzed after excluding 50 HCWs who had undergone previous anti-tuberculosis treatment or LTBI treatment Fig. . We alsoBaseline characteristics of HCWs are shown in Table p < 0.001)  Fig. B. At age01) Fig. B, and th01) Fig. A  vaccination, a TB notification system, and appropriate treatment through public health systems [ARI with ommunity . The mai systems , 13. AddTo date, trends for TB prevalence and ARI have been documented in several countries, and most research has been based on national TST surveys –16. In SOur study also suggested that LTBI was related to older age, healed TB lesion on chest x-ray, and male gender, and not to current occupation in high-risk TB department. Several previous studies reported that TB-related departments such as respiratory ward or clinic, medical ICU, and emergency department increased the risk of LTBI positivity as compared to other departments –24. HoweAlthough the ARI for TB gradually decreased over 20 years in our study, LTBI prevalence and ARI among HCWs in South Korea were still higher than those in developed countries. This study also showed that the prevalence of LTBI was affected more by age-related risk exposure than working in a TB-related department. It is well known that exposure to TB at a young age and recent infection are major risk factors for the progression to active TB , 28. TheThis study has several limitations. First, we conducted this study at a single, tertiary hospital; hence, the findings may not be representative of HCWs in other hospital nor general population in South Korea. Second, in calculating ARI in this study, we used cross-sectional data rather than longitudinal data; hence, the accuracy of findings for old age may decrease given that the IGRA test scores wane as the age increases. Despite these limitations, to our knowledge, this is the first study in South Korea to report the estimation of ARI for TB based on IGRA results, and our study included large HCWs of different ages, working department, and working durations. Considering that age has a great impact on LTBI positivity, long-term follow-up studies are needed to identify how the progression from LTBI to active TB differs by age group.Our study showed that ARI in HCWs in South Korea has gradually decreased over the last two decades, but LTBI was still considerably prevalent. We also suggest that LTBI among HCWs might be greatly affected by age, rather than occupational exposure in intermediate TB burden countries, hence different LTBI treatment strategies by age group should be considered.Additional file 1: Figure 1. Predictive value of Age for IGRA positivity.Additional file 2: Table 1. IGRA positivity according to occupation and working department.


Albizia coriaria Welw ex. Oliver (Fabaceae) is one of the plants used by herbalists in the East Africa community to prepare herbal remedies for the management of symptoms of TB. Despite its widespread use, the antimycobacterial activity of this plant was uninvestigated and there was contradicting information regarding its cytotoxicity. A. coriaria. Gas chromatography-mass spectrometry (GC-MS) followed by Fourier transform infrared (FTIR) spectroscopy was carried out on the acetone and methanol extracts. The binding affinities and descriptors of pharmacokinetics and toxicity of the identified compounds were predicted using computational modelling software.  Cytotoxicity (MTT), antimycobacterial activity (MABA), and phytochemical screening were conducted on crude extracts  of the stem bark of μg/mL. The minimum inhibitory concentration of both the acetone and methanol extracts was 1250.0 ± 0.0 μg/mL against M. smegmatis, whereas that against M. tuberculosis was 937.0 ± 442.0 μg/mL and 2500.0 ± 0.0 μg/mL, respectively. Hexane and chloroform extracts were not active against both strains. Alkaloids, triterpenes, flavonoids, tannins, and saponins were the predominant phytochemicals present. GC-MS analysis revealed twenty-eight and nineteen compounds in acetone and methanol extracts, respectively. Among these was hydroquinone, which was previously reported to possess antimycobacterial activity. Seven compounds identified through GC-MS analysis had better binding affinities for the mycobacterial ATPase and polyketide synthase-13 than isoniazid and rifampicin. These compounds also showed variable but promising pharmacokinetic properties with minimum toxicity.  The cytotoxic concentrations of all extracts were greater than 1000 A. coriaria stem bark with potential antimycobacterial activity and acceptable cytotoxicity, which can be further explored and optimized for the development of novel antitubercular drugs. There are phytochemicals in  Mycobacterium tuberculosis (M. tuberculosis), a highly infectious bacillus that mainly affects the pulmonary system but also other extrapulmonary organs -2, 5 diphenyl tetrazolium bromide (MTT) assay was used to assess the cytotoxicity of the extracts using mammalian Vero E6 cells (ATCC CCL 81) . Cryopre50 value), was estimated from the graph using a linear regression equation (Y = aX + b).The results were used to construct graphs of percentage cell viability against extract concentrations. The cytotoxic concentration of plant extract, which corresponds to 50% viability of the Vero cells (CCM. tuberculosis (H37Rv) and Mycobacterium smegmatis (ATCC 607) were obtained from the TB laboratory at the Centre for Respiratory Disease Research (CRDR), Kenya Medical Research Institute, Nairobi, Kenya. M. smegmatis was used because of its nonpathogenicity, first replicating nature and genetic similarity to M. tuberculosis  have been reported to possess antibacterial activity  . The mosactivity .A. coriaria showed peaks that corresponded to different functional groups present in the respective extract. In the acetone extracts, the peaks at 1027.87 and 1204.32 cm−1 are for C-O bonds, the peak at 1370.17 cm−1 is for C-O-H bonds, peak at 1604.48 cm−1 is for C=C or C=O, and peak 3208.98 cm−1 is for O-H  values of less than 2 Å Table. . An RSMDSeven compounds identified by GC-MS analysis had better binding affinities than the two drugs (Isoniazid and rifampicin), which form the backbone of any first-line TB regimen. Five compounds had better binding affinities than bedaquiline  . A visuaDescriptors of pharmacokinetic and toxicity properties of the top docked compounds were calculated to predict and give an insight into the drug-like properties of the compounds. These parameters can be used to guide future optimization of the promising compounds into leads by focusing on those compounds with optimum ADMET properties. Among the pharmacokinetic descriptors that were calculated included water solubility, human intestinal absorption, plasma protein binding (PPB), blood-brain barrier (BBB) penetration, CYPP450 inhibition, volume of distribution (VD), clearance, half-life, and skin permeation. On the other hand, the toxicity parameters that were calculated included AMES toxicity, hERG blockers, hepatotoxicity, and skin sensitization .A. coriaria contains more polar extractable phytochemicals than their nonpolar counterparts. In a related study, ethanol, a solvent of comparable polarity to methanol had the highest yield in the extraction of phytochemicals from the leaves of A. coriaria [Methanol and acetone had significantly higher yields than hexane and chloroform because of their high and intermediate polarity that allows the extraction of more compounds with intermediate to high polarity. These results are in line with the known behavior that polar solvents extract more compounds than nonpolar solvents. The results, therefore, suggest that the stem bark of coriaria .50 ≤ 20 μg/mL = highly cytotoxic, IC50 21–200 μg/mL = moderately cytotoxic, IC50 201–500 μg/mL = weakly cytotoxic, and IC50 > 501 μg/mL = noncytotoxic [μg/mL. This probably suggests that all the stem bark extracts  of A. coriaria lack inherent toxic phytochemicals that can destroy mammalian cells at normal concentrations. In line with this study, the methanol and water stem bark extracts of A. coriaria were nontoxic (CC50 > 500 μg/mL) against human embryonic lung fibroblast (HEL) cells [A. coriaria harvested in central Uganda had high cytotoxicity against the human glioblastoma cell line (U87.CD4.CXCR4) with CC50 of 6.4 and <4 μg/mL, respectively [A. coriaria is used in the management of TB, which is a chronic disease that develops and heals slowly, subacute and subchronic toxicity studies are needed to determine the long-term effects of the repeated administration of the bioactive plant extracts on hematological, biochemical, and histological parameters [The National Cancer Institute classified chemicals according to their cytotoxicity as follows: ICytotoxic . The cytL) cells . In contectively . The diframeters .A. coriaria [in vitro antimycobacterial activity of A. coriaria. However, other species from genus Albizia such as A. adianthifolia have been reported to possess antimycobacterial activity [The observed bioactivity of the acetone and methanol extracts implies that compounds of intermediate to high polarity are responsible for the inhibition of the growth of mycobacterial species. Although we could not ascertain the exact phytochemicals responsible for the bioactivity at this stage, it is highly plausible that the observed activity is due to compound(s) present in this plant such as triterpenes, alkaloids, and flavonoids. Acacic acid lactone, lupeol, lupenone, (+)-catechin, betulinic acid, and benzyl alcohol were previously isolated from the ethyl acetate extract of the stem bark of coriaria . Additiocoriaria . The inacoriaria . The higactivity .The phytochemicals present are secondary metabolites of various pathways that occur in many plant species of family Fabaceae. Some of these phytochemicals have been reported to possess good pharmacological activities against TB and hence could be responsible for the antimycobacterial properties of this plant . The div2, and -SH groups more volatile, less polar, and more thermally stable. Although BSTFA is a strong silyl donor, its donor strength is increased in the presence of TMCS [Derivatization of less volatile and thermally labile phytochemicals using appropriate derivatizing agents is a method of increasing the detection of phytochemicals during GC-MS analysis. In this study, silylation using BSTFA and TMCS was used to make the phytochemicals with - OH, -COOH, =NH, -NH of TMCS . The derA. coriaria are secondary metabolites that form an integral part of the plants' defense systems. These secondary metabolites have been reported to have significant pharmacological properties and thus used in the management of several human and animal diseases. For example, hydroquinone, which was identified to be present in this plant was previously reported to have antimycobacterial activity with MIC of ranging between 12.5 and 50 μg/mL against susceptible M. tuberculosis (strains H37Ra and H37Rv) and resistant strains [Mycobacterium species with MIC ranging between 12.5 to >100 μg/mL and 6.125 to 25 μg/mL, respectively [Staphylococcus aureus, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus with corresponding inhibition zones of 11, 12, and 9 mm at a concentration of 50 μg/disc [Staphylococcus epidermidis, Streptococcus pneumoniae, Escherichia coli, Micrococcus luteus, Klebsiella pneumoniae, Shigella flexneri, Vibrio cholerae, and P. aeruginosa [A. coriaria has the potential to provide novel molecules for the development of antimycobacterial and antibacterial agents. Apart from acting as marker compounds for bioactivity, the identified compounds can as well be used during chemical fingerprinting and profiling of chemical products especially pharmaceuticals prepared from this plant.All the functional groups that were identified during the FTIR analysis correlated well with the compounds identified during the GC-MS . The maj strains . The samectively . Hydroqu μg/disc . Hexadec μg/disc . Dibutylruginosa –37. Thesα-alkyl, β-hydroxy fatty acids consisting of 60–90 carbon atoms required for the formation of the mycobacterial cell wall and hence maintenance of cell integrity. The interactions of the mycolic acids with other cell wall components make the mycobacterial cell walls very unique and contribute to the challenges associated with mycobacterium especially its pathogenesis, persistence, and chemotherapy. Given the critical role of mycolic acids in mycobacterium cell viability and pathogenesis, enzymes involved in mycolic acids biosynthesis, such as pks-13 present novel targets for drug development [M. tuberculosis ATP synthase is the suppression of the ATP hydrolase activity and its inability to establish a proton gradient. This is thought to be an adaptive mechanism to prevent wastage of ATP during low oxygen conditions [In mycobacterium, polyketide-13 synthase (pks-13) catalyzes the condensation reaction (last step) in the biosynthesis of mycolic acid biosynthesis to form oxomycolic acid intermediate, which is then reduced by mycolyl reductase to form mycolic acid. Mycolic acids are long elopment . On the elopment . A uniqunditions . Bedaquinditions .N-(2-chloroethoxycarbonyl)-N-methyl-, pentadecyl ester blocked the pks-13 and ATP synthase with binding energies of -26.8626 and -28.7742 . In a related study to evaluate the antimycobacterial activity of a series of 3,5-disubstituted-1,2,4-oxadiazole derivatives, pks-13 was used as a putative molecular target [π-π interactions with the aromatic amino acid residues Phe1590, Tyr1637, Phe1585, Tyr1674, Phe1670, and the catalytic His1699 with intermittent hydrogen bonding with Ser1636 and Asn1640 [α-branched aliphatic moieties, which are more effective in suppressing the PMF and thus inhibit the synthesis of ATP [M. tuberculosis [The seven identified compounds that had good binding affinities for the pks-13 and ATP synthase provide insight into their potential to inhibit vital metabolic processes in the mycobacteria. For example, 3′,8,8′-trimethoxy-3-piperidyl-2,2′-binaphthalene-1,1′,4,4′-tetrone and l-leucine, r target . Like in Asn1640 . In anotP), which was violated by compounds 27, 22, 3, 25, and 37 (log P greater than 3). Additionally, all the compounds had moderate water solubility with their log S ranging between −4.9 and −7.8 (optimum solubility ranges between −2 and −4.0). With exception of compounds 43 and 37, the rest had low oral gastrointestinal absorption. This implies that these compounds might need optimization to increase their water solubility and hence absorption as they need to first dissolve in water to be absorbed [43, 3, and 37, the rest were also not inhibitors of the P-glycoproteins. P-Glycoproteins are efflux pumps that actively pump drugs out of the cells, thereby decreasing their intracellular concentrations. They particularly protect the central nervous system from accumulating xenobiotics. Physiologically, P-gp has a fundamental role in the secretory processes [43, which had a skin permeation value of greater than −5 cm/s.All the compounds had acceptable physicochemical properties with acceptable violations of the Lipinski rules except for the logarithm of the n-octanol/water distribution coefficient  and larger molecules (100%) from entering the central nervous system (CNS). However, it allows transport of only water- and lipid-soluble molecules and selective transport of some molecules and drugs especially those that are substrates of active transporters such as glucose and P-gp transporters [All the compounds had high plasma protein binding (greater than 90%) and hence high chances of adsorbing onto the plasma proteins. This might contribute to these compounds having a small therapeutic index and hence high chances of toxicity. The compounds also showed acceptable volumes of distribution. All the compounds had a low fraction unbound in plasma implying that there are less likely to efficiently transverse the cellular membranes and reach the target sites. All the compounds showed the inability to penetrate through the blood-brain barrier except compound sporters .3 showed inhibitory potential on all the cytochrome P450 (CYP 450) enzymes, whereas compounds 43, 27, and 37 showed the inhibition of CYP 2C9 and CYP3A4. CYP 1A2 was inhibited by all the compounds except compound 43. All compounds were substrates to CYP2C9, whereas compound 3 was in addition a substrate for CYP2D6 and CYP 1A62. CYP 450 enzymes are mixed functional oxidases located majorly in the liver and intestine and are responsible for the metabolism of about 60% of xenobiotics such as drugs, steroids, carcinogens, and eicosanoids. They mainly catalyze reductions, oxidation, hydrolysis, and epoxidation reactions that result in the transformation of the parent compound into either less or more toxic metabolites. Compounds that inhibit CYP450 can lead to serious drug-drug interactions that might result in toxicity or pharmacokinetic enhancement. Compounds that are substrates to CYP 450 are most likely to be metabolized, and this can result into either their deactivation or creation of more active metabolites [Compound abolites .25 and 37, the rest of the compounds had low clearance rates implying that they are more likely to accumulate and stay longer in than body. Clearance affects both the half-life and bioavailability of drugs, thereby directly influencing the dose and dosing frequency of a drug. As much as low clearance is advantageous as it leads to less frequent dosing, it might enhance the toxicity of the compound if the toxicity is concentration-dependent [43, which showed mutagenic potential. In conclusion, the ADMET results imply that the top docked compounds possess variable promising pharmacokinetic properties with minimum toxicity. Hence, the most active compounds in silico could be isolated and considered for optimization and further validation.With the exception of compounds ependent . AlthougThe NIST library could have lacked some chemical compounds that were present in the extracts and therefore could not be identified. It is also possible that some less volatile and thermally labile compounds were not derivatized and hence could not be detected by the GC-MS system.A. coriaria with potential antimycobacterial activity and acceptable cytotoxicity, which can be further explored and optimized for the development of new antitubercular drugs.There are phytochemicals in


ACADM variants. We report a case of an asymptomatic Japanese girl with MCAD deficiency caused by compound heterozygous pathogenic variants  and c.449_452delCTGA (p.Thr150ArgfsTer4)). Because the MCAD residual activity in lymphocytes of the patient was below the limit of quantification, both variants are likely to cause complete loss of MCAD enzymatic activity.Medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency is an autosomal recessive disease caused by biallelic pathogenic  ACADM gene (OMIM 607008) variants. In hepatocytes, fatty acid oxidation provides acetyl-CoA for hepatic ketogenesis, which serves as a major source of energy once hepatic glycogen stores are depleted. Because MCAD catalyzes the first step of mitochondrial beta-oxidation for medium-chain acyl-CoAs, a previously healthy individual with MCAD deficiency (MCADD) suddenly experiences symptoms triggered by periods of fasting or illness, such as viral infection. Symptoms include hypoketotic hypoglycemia and vomiting, which may quickly progress to lethargy, seizure, and coma. Sudden unexpected death in infancy (SUDI) has also been recognized as the first manifestation of MCADD2. It is reported that before newborn screening (NBS) was available, 20–25% of affected infants and young children died suddenly during the first episode of metabolic decompensation and that severe neurological sequelae were often observed in survivors3. In contrast, once the diagnosis is established, implementation of frequent feeding to avoid any prolonged fasting and appropriate management of periods of illness can be expected to almost completely prevent onset of the disease5.Medium-chain acyl-coenzyme A dehydrogenase  deficiency (OMIM 201450) is the most common disorder of fatty acid beta-oxidation caused by biallelic pathogenic 7, which is considerably lower than that in Western countries. In 2014, Japan initiated a nationwide NBS for MCADD using tandem mass spectrometry (MS/MS), which facilitated the identification of many patients and novel variants. Although it is important to evaluate the risk of acute metabolic decompensation for each variant, a lack of knowledge about the association between variants and enzyme activity makes it difficult to infer phenotype from genotype. Here, we report a Japanese girl with MCADD with an unknown pathogenicity ACDM gene variant.The prevalence of MCADD varies across ethnic groups. In Japan, it is estimated to be approximately 1 in 100,000 birthsACADM gene was performed, and we identified two heterozygous mutations: NM_000016.5:c.449_452delCTGA (p.Thr150ArgfsTer4) and c.1040G > T . Targeted Sanger sequencing identified c.449_452delCTGA and c.1040G > T in her father and mother, respectively , 1000 Genome Database (http://browser.1000genomes.org/), dbSNP (http://www.ncbi.nlm.nih.gov/snp/), Genome Aggregation Database , Human Genetic Variation Database , and Japanese Multi Omics Reference Panel . Moreover, this missense variant is predicted to be probably damaging, with a score of 0.979, by PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/) and deleterious, with scores of −8.147 and −5.25173, by PROVEAN (http://provean.jcvi.org/index.php/) and PANTHER (http://www.pantherdb.org/tools/csnpScoreForm.jsp/), respectively. Although c.1040G > T has very recently been reported as a novel variant observed in 1 of 24 Chinese patients with MCADD9, no detailed assessment was performed to classify the pathogenicity of this variant with evidence according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines10. This missense variant is absent in the population database (PM2); it may cause a deleterious effect on the gene product, as supported by multiple lines of computational evidence (PP3). It is detected in trans with a pathogenic variant (PM3) and was observed in the gene specifically associated with the patient’s phenotype (PP4). Consequently, this variant is classified as ‘likely pathogenic’ according to ACMG guidelines10. We measured the enzymatic activity of MCAD using the patient’s lymphocytes11, and residual activity was 0% of normal (below the limit of quantification); thus, both variants appear to cause complete loss of MCAD function . She was discharged from the maternity clinic at 5 days of age, with no special remarks regarding her perinatal history. Because elevated octanoylcarnitine (C8) levels and C8/decanoylcarnitine C10) ratios (C8/C10 ratios) were noted by NBS, she was referred to our hospital. At the first visit, no abnormalities were observed on physical, neurological, or blood examination. Results of NBS at 5 days of life were 39.453 nmol/ml, 4.027 nmol/ml , and 8.628  for free carnitine (C0), C8, and C8/C10 ratios, respectively. Urinary organic acid analysis at 7 days of life detected 2.9 nmol/ml hexanoylglycine  and 12.4 nmol/ml suberylglycine . Although the findings of urinary organic acid analysis in this disease are not specific and serum acylcarnitine analysis is recommended as a basis for diagnosis, serum acylcarnitine analysis was not feasible in our hospital. Therefore, we suspected MCADD based on the results of acylcarnitine analysis of dried blood spot samples and urinary organic acid analysis and performed enzyme activity analysis and genetic analysis for a definitive diagnosis. After written informed consent was obtained from her parents, next-generation sequencing (NGS) of the patient’s 0 ratios ACADM gene missense variant, NM_000016.5:c.1040G > T, in a patient with NBS-positive asymptomatic MCADD. The complete loss of MCAD activity in the patient’s lymphocytes indicates that those with compound heterozygous c.1040G > T  and c.449_452delCTGA (p.Thr150ArgfsTer4) mutations are at high risk of metabolic decompensation.In conclusion, we identified a pathogenic The relevant data from this Data Report are hosted at the Human Genome Variation Database at 10.6084/m9.figshare.hgv.3112.


Parus major) from nine European cities, each paired with an adjacent rural site, to address this major knowledge gap in our understanding of wildlife urban adaptation. We find that a combination of polygenic allele frequency shifts and recurrent selective sweeps are associated with the adaptation of great tits to urban environments. While haplotypes under selection are rarely shared across urban populations, selective sweeps occur within the same genes, mostly linked to neural function and development. Collectively, we show that urban adaptation in a widespread songbird occurs through unique and shared selective sweeps in a core-set of behaviour-linked genes.Urbanisation is increasing worldwide, and there is now ample evidence of phenotypic changes in wild organisms in response to this novel environment. Yet, the genetic changes and genomic architecture underlying these adaptations are poorly understood. Here, we genotype 192 great tits ( The genetic architecture underlying rapid adaptive responses to novel environments are poorly understood. A study of great tits from nine European cities finds that urban adaptation in a widespread songbird occurred through unique and shared selective sweeps in a core-set of behaviour-linked genes. Many species show phenotypic adaptations to the multiple urban challenges, such as higher levels of noise, artificial light at night, air pollution, altered food sources or habitat fragmentation6. These adaptations include changes in behaviour, e.g., refs. 9, morphology and locomotion, e.g., refs. 13 or toxin tolerance14. Indeed, there is now evidence that the phenotypic divergence between urban and rural populations may have a genetic basis in some species15, in line with the finding that micro-evolutionary adaptations in natural populations can occur within short timescales, particularly in response to human activities18. The study of the genetic signals of adaptation could provide important insights into the magnitude of the evolutionary change induced by urbanisation on wildlife. However, the short evolutionary timescale, the dependence of evolution on local factors, and the polygenic nature of many phenotypic traits, make detecting the genomic signals of adaptation difficult19.Urban development is rapidly expanding across the globe, and although urbanisation is regarded a major threat for wildlife20 or on a narrow geographical scale, e.g., refs. 22, thereby limiting the inferences that can be made on the consistency of genomic responses to urbanisation5. Conversely, the study of multiple populations on a broad geographical scale provides a powerful framework to identify the evolutionary forces shaping the genomic responses and to test the repeatability of urban adaptation24. In particular, comparing across populations enables the distinction between random demographic non-adaptive processes, such as genetic drift, and signatures of natural selection, as selection might be expected to affect the same genomic regions (nucleotides or genes) and/or functional pathways across cities25. The current evidence on the genomic basis of urban adaptations ranges from high parallelism, involving a single or few genes23, to polygenic, and to putative genetic redundancy, e.g., ref. 22. This discrepancy might be due to the variable and sometimes local urban selection pressures together with the polygenic nature of many adaptive phenotypic traits, which could lessen the likelihood for shared genetic changes at the nucleotide level26.The majority of studies on the genomic basis of urban adaptation have either focused on a limited number of markers and genesParus major), to identify the genetic basis of urban adaptation. The great tit is a widely distributed songbird and a model species in urban, evolutionary and ecological research, e.g., refs. 36, with demonstrated phenotypic changes in response to urban environments in several populations37. In addition, genomic resources are well developed for this species38 and it is known that across its European range, the species presents low genetic differentiation41. In order to examine genomic responses to urbanisation on a broad geographical scale, we analyse pairs of urban and rural great tit populations from nine localities across Europe .A total of 192 great tits from the nine paired urban–rural populations were genotyped at 517,603 filtered SNPs, with 10–16 individuals per sampling site  except for the urban populations in Munich and Paris  within population pairs , as well as between urban populations (urban vs urban), was on average higher than the differentiation between rural populations  across Europe  compared to rural populations  showed that urban birds likely independently colonised multiple times European cities from forest sources42. In our study, the finding of independent clusters for some urban populations also point towards a similar scenario, i.e., an independent and repeated colonisation of urban habitats from largely admixed rural populations. However, the overall weak genetic divergence and the detection of significant migration events across distant populations suggests a possible role of gene flow in the facilitation of urban adaptation, via the spread of adaptive alleles. Still, in our subsequent analyses we treated all our urban–rural population pairs as independent, including Munich and Paris, as selection pressures might differ across cities and result in idiosyncratic genomic responses to selection despite any ongoing gene flow.Overall, our results reveal weak population structuring across Europe, with slightly increased genetic differentiation between urban populations compared to the more admixed rural populations. Previous results in another European songbird, the blackbird  and those with more genes , based on all urban-associated SNPs detected by LFMM, clearly separated urban and rural populations along PC1GEA  approaches, LFMM (latent-factor mixed models) and an additional Bayesian approach (using BayPass). Testing for GEAs with LFMM  is higher compared to the effect size of a significant “habitat × locality” interaction term, which describes differences in the direction and magnitude of the allele frequency change between cities44. Applying these models to each urbanisation-associated SNP from the LFMM model (2758 SNPs), we detected large variation in allele frequency shifts across localities . In contrast to this, most of the “core urbanisation SNPs” showed similar allele frequency differences across localities, with 76% of them showing a main effect of the urban habitat  and the “core urbanisation SNPs” . In comparison, both the effect of “locality”, which corresponds to the distinct evolutionary history of the local populations , and the interaction of “habitat × locality” , explained much smaller proportions of the total variation  than the habitat term by itself. In contrast, genetic variation along PC2 and PC3 was mostly inconsistent across localities and likely specific for each city  on allele frequency shifts in the detected SNPs, we assessed using univariate linear models, the explanatory power of habitat, locality (city), and their interaction on the direction and strength of allele frequency shifts per-SNP across all paired populations. A significant “habitat” term indicates consistent allele frequency shifts across cities, particularly when the effect size  and white-footed mice (Peromyscus leucopus)45. Therefore, we further performed genome-wide scans of differentiation and selective sweep analyses in each of our studied populations to test if these contributed to the urban adaptation in great tits and, importantly, to explore if selective sweeps were population specific or widely shared across Europe.While GEA approaches are powerful tools for identifying even subtle allele frequency shifts associated with local adaptationXP-nSL)46. Using this approach, we found that between 436 and 700 genomic windows , which clustered into 127–173 wider genomic regions, showed signatures of selection in urban populations  or geographic distance , suggesting that shared adaptive genetic variation through introgression and gene flow between neighbouring urban populations likely did not facilitate urban adaptation. However, we cannot fully exclude the role of gene flow as migration was generally strong, even across large distances  between each adjacent urban and rural pair across the same windows  were largely population specific  and clustered genomic outlier regions  compared to XP-nSL. This result suggests that ongoing/recent sweeps outnumber older and completed selective sweeps in urban great tits. Similar to XP-nSL, the Rsb outlier windows were only shared across a maximum of five populations and in general widely distributed across the genome , were pervasive across the genomes of urban great tits, supporting our inference of a highly redundant polygenic adaptation to urban environments. The underpinning genomic changes associated with urban adaptation were largely population specific, likely the result of differential selective pressures, adaptation through complex multivariate phenotypic changes and/or selection on standing genetic variation50. Nonetheless, we still detected shared selective sweeps across urban populations, suggesting the presence of common genomic responses to the urban selective pressures, either through repeated selective sweeps or sharing of adaptive genetic variants.Overall, the selection analyses showed that selective sweeps, and in particular those that are recent and ongoing  and differentiation (FST) with two proxies of genomic recombination rate, i.e., LD and intronic GC-content (both in 200 kb non-sliding windows). LD is correlated with population-specific recombination rates and the strength of selection, while intronic GC-content is a surrogate for the long-term recombination rate variation across the great tit genome, i.e., a higher GC-content will indicate a higher recombination rate52. To confirm that both proxies provide similar pictures of the recombination landscape, we tested their correlation across all population pairs. Indeed, LD and GC-content, were (negatively) correlated in all populations , and 362–923 with completed selective sweeps (Rsb). Of these genes, 64–207, and 5–76, were detected in at least two urban populations . Such consistent genomic responses on the gene or functional level could explain the similarity in phenotypic responses to environmental variation despite a large variation on the haplotype levelχ21 = 77.95, P < 2.2 × 10−16), we conservatively focused our functional interpretation only on those genes that were associated with signatures of selection (XP-nSL or Rsb) in more than half of all urban populations (≥5 urban populations), as these likely play more important roles in urban adaptation. In total, we detected 42 genes associated with recent or ongoing selective sweeps in at least five populations (XP-nSL: max. six populations), and 15 genes associated with older completed selective sweeps  or genetic distance , which is in accordance with the results regarding shared outlier windows and further supports that many selective sweeps likely occurred independently in multiple urban populations. In addition, 12 of the 57 shared genes under selection were also associated with urbanisation in the LFMM analysis  in seven and six populations, respectively, but also previously shown to be differentially expressed in blood and/or liver between urban and rural birds from Malmö59. Moreover, following Malmö as an example, one gene found to be differentially expressed in Watson et al.59, VPS13A, was highly differentiated (ZFST) and associated with selective sweeps in that particular population, but importantly, also under selection in Madrid (Rsb) and Lisbon (XP-nSL) and in general, associated with urbanisation . This exemplifies that some genes show signatures of urban adaptation on a continental scale, making them strong candidates for adaptation in these urban centres.Although it is statistically unlikely to detect selective sweeps in the same gene in more than three urban populations by chance alone , the exact locations of these signatures were not consistent  codes for a receptor of the neurotransmitter serotonin, a pathway consistently identified as target of changes linked to urbanisation. Indeed, two previous studies in birds, one on European blackbirds using a candidate gene approach and another on burrowing owls (Athene cunicularia), using full-genome resequencing, also described changes in this pathway as the main difference between urban and rural populations22. Both species also show consistent urban–rural differences in two behaviours associated with serotonin: down-regulation of the stress axis and altered nocturnal behaviour under artificial light at night, respectively62. The same gene, HRT7, has also been directly linked to behavioural differences between migratory and resident rainbow trout (Onchorynchus mykiss)63, supporting its potential role in behavioural changes in urban great tits. The CDH18 gene (Chr 2), is part of a superfamily of membrane proteins involved in synaptic adhesion and revealed as a candidate gene in phonological alterations in humans64. The PTPRD and VPS13A genes (both in Chr Z), are suggested to be involved in hippocampal neural development65 and were previously linked to bird navigation, flight performance and migratory behaviour68. Moreover, two other genes under selection and associated with urbanisation are involved in the regulation of cognitive processes and behavioural disorders in vertebrates (DLG2 (ref. 69) and NRXN3 (ref. 70), Chr 1 and 5, respectively), and were also previously linked to urban divergence in burrowing owls22.Many of the genes associated with urbanisation have previously been linked to behavioural divergence, and cognitive and learning functions, suggesting adaptive phenotypic shifts related to behaviour. In particular, 72. These findings were furthermore supported by gene ontology (GO) analysis of the 2758 urbanisation-associated SNPs , linked to 984 genes (1501 SNPs in genic regions). Accordingly, most of the GO terms were related to neural functioning and development , cell adhesion  and sensory perception . Each of the paired sampling sites  was sampled within the same season. Barcelona and Munich were sampled during winter, however, in both cases only known birds (recaptures) were included in the study, thus, all birds can be considered resident. All urban populations were located within the city boundaries, the areas are characterised with significant proportion of human-built structures, such as houses and roads with managed parks as the only green space  were obtained either from the jugular or brachial vein and stored at 4 °C in ethanol or SET buffer and subsequently frozen at −20 °C. In each case, procedures were identical for the paired urban and rural populations. All procedures employed during field work were approved by national Ethical Committees and authorisations to collect samples were delivered by the Environment Department of the Generalitat de Catalunya (permit no. AECC/SF/0438), the Scottish Natural Heritage (permit no. 52463) and UK Home Office (license no. 70/7899), the Malmö-Lund animal Ethical Committee (permit no. M454 12:1), the CEMPA and Portuguese Ministry of Environment (permit nos. 40/2014 and 164/2014), the Ministry of the Environment, Housing and Territorial Planning of Madrid , the Institute for Environmental Protection and Research  and the Lombardy Region (permit no. 3462), the Tierschutzgesetz  and the Regierung von Oberbayern (permit nos. 55.2-1-54-2532.2-7-07 and 55.2-1-54-2532-140-11) and the Minister of Higher Education, Research and Innovation , the Prefect of Paris and the Prefect of Seine et Marne (permit nos. DRIEE-2012-31 and DRIEE-2012-32) and the CRBPO . DNA was extracted from ~5 µl samples of red blood cells in 195 µl of phosphate-buffered saline, using Macherey-Nagel NucleoSpin Blood Kits , and following the manufacturer’s instructions or manual salt extraction (ammonium acetate). The quantity and purity of the extracted genomic DNA was high as measured using a Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific) and Qubit 2.0 Fluorometer (Thermo Fisher Scientific).75, based on aerial images from Google Maps (Google Maps 2017), and following the methods described in different studies assessing the effect of urbanisation on wild bird populations76. Briefly, each sampling site was represented by a 1 × 1 km rectangular area around the capture locations. We opted to use this spatial resolution around the sampling sites to approximately cover individuals’ territory32, but also to ensure that we considered the heterogeneity within each selected site. The content in each rectangle was evaluated dividing the image in 100 × 100 m cells and considering three land-cover characteristics in each: proportion of buildings, vegetation (including cultivated fields) and paved surfaces  for each of the urban or rural populations per locality, see Table urb values were transformed to obtain negative values in the less urbanised and positive values in the more urbanised sites. We used the average of the urbanisation estimates if birds were captured in more than one location within each site . All quantifications were done in triplicates by the same person (P.S.) and the estimates were highly repeatable  = 0.997–0.987, P < 0.001).To quantify the degree of urbanisation at each site, we used the UrbanizationScore image analysis software38. Thus, this SNP chip allowed us to genotype a large number of individuals on a genome-wide basis at high SNP density without a strong ascertainment bias. SNP calling was done following the “Best Practices Workflow” in the software Axiom Analysis Suite 1.1.0.616 (Affymetrix©) and all the individuals passed the default quality control steps provided by the manufacturer  and previous studies using the same SNP chip77. A total of 544,610 SNPs were then exported to a variant-calling format (VCF) and Plink v. 1.9 (ref. 78), and further filtered and assigned to chromosomes using the P. major reference genome build 1.1 . A total of 155 SNPs were not found in the used assembly and 26,852 SNPs were not in chromosomic regions; thus, these SNPs were removed from further analysis leaving a total of 517,603 SNPs.All 192 individuals were successfully genotyped using a custom made Affymetrix© great tit 650 K SNP chip at Edinburgh Genomics . The Affymetrix SNP chip was developed based on whole-genome sequencing data from great tits sampled across multiple European populations, largely corresponding to our sample sitesHe) for each population using Plink v. 1.9 (ref. 78), and tested if genetic diversity significantly differed between urban–rural populations from the same location using t tests in R, and overall across urban–rural pairings using a Wilcoxon rank-sum test in R v.3.6.1 (ref. 79). Furthermore, we estimated pairwise FST between all population pairs , using VCFtools v. 0.1.15 (ref. 80). Mean average FST was computed across all comparisons after setting negative values to zero.We calculated the genome-wide genetic diversity as expected heterozygosity (78), using a variance inflation factor threshold of 2 (“-indep 50 5 2”), retaining 358,149 SNPs. Using this LD-pruned dataset, we performed a PCA using Plink. We performed two different PCAs, (i) only with autosomes and excluding all small linkage groups and the Z-chromosome, and (ii) including the Z-chromosome but excluding all small linkage groups. Subsequently, we performed additional dimensionality reduction for each PCA dataset using the first 20 principal components, using the UMAP v.0.2.7.0 R-package with default settings81. We also compared the results to the UMAP based on the first ten PCs. Genetic ancestry analysis was performed using the software package fastStructure v.1.0 (ref. 82), with K ranging from 2 to 9 and cross-validation. In addition, we inferred a population tree based on allele frequency co-variances using Treemix v.1.3 (ref. 83), with blocks of 100 SNPs. We fitted up to five migration edges and determined the optimal number of migration edges that lead to the best fitting population tree by (i) comparing the likelihoods of trees with different number of migration edges, (ii) estimating the total variance explained by each tree and (iii) by comparing the significance of the fitted migration edges.For analyses of population structure, we pruned the SNP dataset based on LD in Plink v.1.9  inferred with fastStructure v.1.0, we ran LFMM with two and four latent factors, respectively. Each model was run five times for 10,000 iterations with a 5000-iteration burn-in. We calculated the median z-score for each locus across all ten runs, and selected SNPs with a FDR < 1% to be associated with urbanisation. The results with two or four latent factors were highly concordant and the same candidate loci were recovered; thus, we only used the results obtained with four latent factors for further analyses. We also assessed the distribution of P values to check for the impact of confounding factors , we detected a far higher number of associated SNPs . We estimated the allele frequency–environment association for each SNP with the urbanisation score for each population accounting for population structure, using a covariance matrix. We estimated the covariance matrix using the LD-pruned SNP dataset in the core model using default parameters: 20 pilot runs of 1000 iterations, a run length of 50,000 iterations, sampling every 25th iteration, and a burn-in of 5000 iterations. The resulting covariance matrix was used as input for five replicated runs of the auxiliary covariate model using the above settings. The strength of association is given in the test by estimated Bayes factor . We calculated the median Bayes factor across all five replicated runs and considered all SNPs with a deciban unit (dB) > 20 as urbanisation associated. This is the strictest criterion and is considered as “decisive evidence” for the association87. Using a resampling-without-replacement-based permutation approach and a chi-square test, we determined if the overlap between the LFMM and BayPass candidate SNPs is higher than expected by chance.Second, we analysed associations with urbanisation using the auxiliary covariate model implemented in BayPass v.2.1 (ref. FST88) for each urban and rural pair for each SNP, using VCFtools v. 0.1.15 (ref. 80). We subsequently summarised and plotted FST values in 200 kb sliding windows with 50 kb steps, using the WindowScanR v.0.1 R-package (https://github.com/tavareshugo/WindowScanR), and standardised window-based FST estimates using a z-transformation (ZFST). Standardisation was performed separately for autosomes and the Z-chromosome.To identify genomic regions distinguishing adjacent urban and rural great tits, we estimated the genetic differentiation . For computational reasons, the analysis was performed on a thinned SNP dataset , for which we randomly selected one SNP every ~50 kb.To determine if the extent of genetic differentiation between all possible population pairs was significantly different from zero, we estimated Rsb score below) to identify genomic regions showing signs of older and completed selective sweeps. We used fastPHASE v.1.4 (ref. 90) to reconstruct haplotypes and impute missing data independently for each chromosome using the default parameters, except that each individual was classified by its population (“-u” option). We used ten random starts of the EM algorithm (“-T” option) and 100 haplotypes (“-H” option). The fastPHASE output files were analysed using rehh v.2.0 (ref. 91) to calculate Rsb statistics per focal SNP. The Rsb score is the standardised ratio of integrated EHHS  between two populations91. This statistic measures the extent of haplotype homozygosity between two populations and follows the rationale that if a SNP is under selection in one population compared to the other, the region around this locus will show an unusually high level of haplotype homozygosity compared to the neutral distribution. In accordance to Gautier and colleagues91, we considered significant SNPs putatively under selection in urban populations based on a threshold of Rsb ≥ 4. To identify genomic regions consistently showing strong signals of selection, we summarised Rsb scores in 200 kb sliding windows (50 kb steps) and selected windows under selection as those with (i) an average Rsb scores above the 95th percentile of the genome-wide distribution (separately for autosomes and the Z-chromosome) and (ii) a proportion of urban outlier SNPs (Rsb > 4) above the 95th percentile of the genome-wide distribution (separately for autosomes and the Z-chromosome). The proportion of outlier SNPs per window was the number of SNPs with Rsb values >4 compared to the total number of SNPs per window92. Because recombination rates can be assumed to be conserved between closely related urban and rural populations, the cross-population comparative nature of the Rsb statistic provides an internal control that cancels out the effect of heterogeneous recombination across the genome48.To identify genomic regions showing signs of selective sweeps in urban–rural population pairs, we used two haplotype-based selection scans. First, we scanned the genome for regions showing differences in extended haplotype homozygosity (EHHS) between urban and rural populations (see nSL93 statistic (XP-nSL) that tests for signatures of ongoing or recently completed selective sweeps and is implemented in selscan v.1.3.0 (ref. 46). XP-nSL contrasts nSL, the number of segregating sites by length, between two populations and thus tests for differential local adaptation. Analogous to Rsb, we conservatively detected genomic regions by a 200 kb sliding windows as those with (i) average XP-nSL scores above the 95th percentile of genome-wide distribution (separately for autosomes and the Z-chromosome) in each population and (ii) proportions of outlier SNPs (XP-nSL > 2) above the 95th percentile. We also plotted haplotype patterns around candidate genes using the haplostrips software v.1.3 (ref. 94).Second, we used a cross-population XP-nSL and Rsb) into outlier regions, if windows were not >200 kb apart, using csaw v.3.12 (ref. 95).To determine larger genomic regions associated with signals of selection, we merged adjacent outlier windows for each selection statistic (n genes (n = number of genes with signatures of selection in each urban population) for each population from the list of all SNP-linked genes using the “resample” function in R, assessed the amount of overlap between populations (from two to eight populations) and repeated the sampling 100,000 times for each comparison. We then calculated the mean and 95% CI for each comparison and compared the number of observed shared candidate genes to the expected number of candidate genes. The expected number of genes showing signs of selection in three or more populations was zero, thus we focused on genes showing signs of selection (Rsb and XP-nSL) in three or more populations.To determine the consistency of selection across urban centres, we implemented a resampling approach to assess the likelihood of genes showing signs of selection in two, three, four or more populations. We resampled with replacement Y ∼ habitat × locality × habitat × locality”) to quantify the effects of (i) “habitat” , (ii) “locality”  and (iii) “habitat × locality” interaction 96. We performed this analysis for all individual SNPs  and based on PC scores from the first three PC axes for all LFMM candidate SNPs (PCLFMM), and those overlapping between the LFMM and BayPass analyses . The PCA was estimated for each SNP dataset using SNPrelate v.3.12 (ref. 97). We used the “EtaSq” function implemented in BaylorEdPsych v.0.5 (ref. 98) to extract the effect sizes  for the model terms in each linear model.To determine the explanatory power of urbanisation-associated SNPs and consistency in allele frequency changes across populations, we used linear models .To estimate the impact of variation in recombination rate and linked selection in low-recombination regions on patterns of divergence , we estimated the correlations of LD and intronic GC-content in 200 kb windows with measures of selection/differentiation (r2) across the genome using Plink 1.9 (ref. 78) for pairs of SNPs located up to 200 kb apart for each urban population . LD estimates were averaged in 200 non-sliding windows using the WindowScanR v.0.1 R-package.First, we estimated patterns of LD (P. major) reference genome build 1.1 , as low intronic GC-content is a good proxy for long-term reduced recombination in that region52. Intron coordinates were extracted from the great tit reference genome using the plyranges v.3.12 (ref. 99) and GenomicRanges v.3.12 (ref. 100) R-packages, and the GC-content for each intron inferred using the “nuc” function in BEDtools v.2.28 (ref. 101). Intronic GC-content values were further averaged across 200 kb non-sliding windows using WindowScanR.Second, we estimated the GC-content for all introns across the great tit  across all 200 kb windows for each population, using the “cor.test” function in R.Lastly, we estimated the genome-wide Pearson correlations between LD, GC-content and all estimators of selection (P. major) reference genome build 1.1 . We used all genes containing SNPs associated with urbanisation . To analyse the enrichment of functional classes, we identified overrepresented GOs , using the WebGestalt software tool102. The gene background was set using annotated great tit genes (annotation release ID 101) containing SNPs from the SNP chip and with Homo sapiens orthologues. H. sapiens genes were used as a reference set as human genes are better annotated with GO terms than those of any avian system 39. We focused on non-redundant GO terms to account for correlations across the GO graph topology and GO terms as implemented in WebGestalt102. An FDR < 0.05 was used as a threshold for significantly enriched GO terms. Furthermore, we searched the public record for functions of individual candidate genes. We also used GOrilla103 to visualise the connections of GO terms associated with LFMM candidate genes and Cytoscape v.3.6.1 (ref. 104) to visualise the GO network and identify all enriched GO terms , including redundant terms.We obtained the gene annotations for all candidate SNPs from the great tit (XP-nSL or Rsb outlier windows. Overlaps between outlier windows and annotated genes were assessed using the plyranges R-package v.3.12.Candidate genes associated with signatures of selection were those that overlapped with significant Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary