Cognitive function was evaluated by the Mini-Mental State Examination (MMSE). Linear regression and a binary logistic regression model were used to explore the relationship between THs and cognition in euthyroidism (TSH level of 0.27 ~ 4.20mIU/L). A total of 957 euthyroidism individuals were included in this study, with a mean (SD) age of 71.34 (6.35) years. In individuals with euthyroidism, serum TSH and FT3 levels were positively associated with cognition ; and serum FT3 and TSH levels were significantly associated with cognitive domains (P < 0.05). Further, euthyroid individuals in the lowest serum FT3 quartile had a twofold increased risk of cognitive impairment compared to those in the highest quartile after adjusting for potential confounding factors. These findings suggested that low levels of FT3 could be an independent risk factor for cognitive impairment in older euthyroid adults. Additionally, a positive linear association exists between serum FT3 levels and cognitive domains . Further studies are needed to determine the underlying mechanisms and the community significance of these findings.Accumulated evidence showed that thyroid diseases induced cognitive decline. However, the relationship between thyroid hormones (THs) and cognition in older euthyroid people is still unclear. Our study aimed to estimate the association between THs within the euthyroid range and cognition in community-dwelling older adults in China. Data were extracted from a cohort study on the health status of rural older adults from the Guizhou province in China (HSRO). Serum thyroid-stimulating hormone (TSH), free thyroxine (FT No treatment is available to slow or stop the deterioration of brain cells in AD. The increasing prevalence of AD will lead to a huge burden for society and families1. Early detection of risk factors and incidence is a critical strategy in the prevention of AD. Thyroid hormones (THs) are essential for the development of the central nervous system (CNS) and for regulating metabolism, neurogenesis, myelination, and cellular repair in the brain throughout the lifespan of an individual4. Both in vitro and in vivo thyroid function studies have indicated that abnormal THs levels could lead to impaired cognitive function6. Studies derived from clinical institutions and patients also showed an association between thyroid function and cognitive function10.Due to the aging population, the incidence of Alzheimer’s Disease (AD) will increase to over 100 million people worldwide by 2050, which implies that 1 in every 85 individuals in the world may suffer from the disease11. Similarly, a collaborative project in 25 cohorts did not support the association between thyroid dysfunction and impairment in cognitive function12. A cohort study found that thyroid disorders were associated with AD development13. Observational studies have yielded inconsistent results, which might be attributed to the unstable manifestations of that thyroid disorders among older adults. Some of these studies were limited by heterogeneity in definitions of thyroid dysfunction, while in others, subclinical thyroid dysfunction in older adults might return to normal over time14. Meanwhile, researchers have also studied the impact of changes in the levels of THs within the normal reference range on cognitive function, particularly in older adults. Such data could provide new insights into the association of levels of THs with cognitive decline15.Nevertheless, some observational studies on individuals in the community have yielded controversial results in older adults. A prospective investigation showed no significant association between subclinical thyroid d ysfunction and cognitive decline4)) within the normal reference range18. However, a cohort study by Samuels et al.19 discovered that minor thyroid hormone variations did not affect the cognition of community-dwelling older adults. Notably, these investigations primarily focused on examining the influence of a single thyroid indicator on cognitive function. Owing to the way the THs affect each other via the hypothalamus-pituitary-thyroid (HPT) axis, there is a lack of comprehensive exploration of the relationship between THs and cognitive function in community-dwelling older adults. Hence, it is necessary to observe the association between thyroid hormones in the euthyroid range and cognitive function in older populations. In this study, we used data from cross-sectional studies to investigate the aging population in a community in Guizhou province.Some studies have found an association between cognition and thyroid indicators (thyroid-stimulating hormone (TSH) or free thyroxine (FTP < 0.001). Further, we observed that MMSE scores significantly declined with increasing age (P < 0.001). Participants with hypertension, depression, or anxiety, and those who were single or widowed/divorced had significantly lower MMSE scores (P < 0.001). Moreover, individuals from currently smoking and currently drinking groups got significantly higher MMSE scores (P < 0.001). Likewise, serum levels of TSH and THs differed by age, gender, and education level, as shown in Table A total of 1235 subjects were included in this study for thyroid function assessment. Of these, 957 (77.49%) individuals had euthyroidism. The demographic characteristics and MMSE scores of the euthyroid participants are shown in Table 3 concentrations. Individuals with lower FT3 concentrations had lower MMSE scores. The quartiles for the TSH concentration group showed statistically significant differences in MMSE scores for concentrations in Q3 and below. Further, we assessed the correlations between various thyroid markers and cognitive performance in different thyroid statuses, revealing that serum FT3 concentration was independently associated with cognitive function in euthyroidism  and TSH  were associated with the MMSE score. Further subgroup analysis revealed that in the TSH levels (Q4) group, FT3 was associated with MMSE scores, while in the high FT3 levels (Q4) group, TSH was not statistically associated with MMSE scores. Moreover, as shown in Fig. 3 was an independent risk factor for cognitive impairment after adjusting for age, gender, anxiety, depression, education level, and other confounding variables. The distribution of characteristics of all subjects assessed for thyroid function and the proportion of cognitive impairment in different groups are shown in Supplementary Tables 3 levels were associated with the cognitive domains of immediate memory and language, while serum TSH levels were found to have a weak positive association with immediate memory and attention , there is an observed association between FT3 and MMSE scores. Conversely, when FT3 levels are high (Q4), TSH is not found to be statistically associated with MMSE scores. The relationship between FT3 and TSH in older adults with euthyroidism involves a dynamic interplay. Low FT3 concentrations may lead to increased TSH secretion, and changes in TSH levels can also influence FT3 concentrations. The findings suggested that the influence of thyroid hormones on cognitive function may vary based on hormone concentrations. Future research should explore the temporal dynamics and bidirectional influences within the hypothalamus–pituitary–thyroid axis and their specific impact on cognitive function.Previous studies have primarily focused on exploring the relationship between thyroid dysfunction and cognition. However, thyroid dysfunction has been found to be less prevalent in older communities, which might be attributed to the heterogeneity in definitions of thyroid dysfunction3 was linked to a twofold risk of cognitive impairment. These findings were in line with a single-center study that evaluated patients with dementia10, which may be attributed to the altered activity of deiodinase type 2 (D2), expressed in various tissues, which regulates the conversion of T4 to T3 in the central nervous system. D2 activity could be considered as another possible mechanism contributing to the association between thyroid hormones and cognitive decline. Further investigations are warranted to delve into this mechanism and establish a more comprehensive understanding. Similarly, our results align with cohort studies that found no correlation between normal range or mild changes in TSH and cognitive dysfunction26. This concordance may be attributed to our study population, consisting of elderly individuals with normal thyroid function. In post-mortem and mono-center studies, AD patients have been found to exhibit low serum levels of FT3, which was consistent with our findings25. Similarly, in older euthyroid adults, a previous cross-sectional study found that the risk factors for cognitive impairment were positively associated with FT3 levels27. Our study contributes to the growing evidence associating FT3 levels with cognition in older euthyroid adults, suggesting a nuanced interplay between TSH and FT3 within the normal thyroid function range. Furthermore, we also found that TSH and FT3 levels were associated with the cognitive domain of immediate memory. Previous observable research found an association between TSH levels in the normal range and memory performance29. The prefrontal cortex is a brain region of great importance for memory30. A study on older mice with significantly impaired learning and memory showed lower serum levels of FT3 and higher levels of SNAP-25 and MUNC18-1 in the frontal lobe of the mice31. This finding held potential significance in enhancing our understanding of the mechanisms linking TSH and FT3 levels with cognitive domains in euthyroidism.In our study, we observed no significant association between thyroid-stimulating hormone levels and cognitive impairment, and the lowest concentration of FT3, total T4, thyroid-specific antibodies, and enzymes involved in the conversion of T4 to T3 might have limited the interpretation of our findings. There is a need for a more comprehensive assessment to elaborate on the mechanisms underlying the impact of thyroid hormones on the central nervous system. Fourthly, the MMSE is a brief assessment of overall cognitive function and may not be sensitive to the more subtle changes across our lifespan.The current study had several limitations. Firstly, this is a cross-sectional epidemiological survey in rural Guizhou province, with no evidence of age-related cognitive changes in the selected cohort. Secondly, this study lacks the exploration of inflammatory markers. All the associations might have arisen due to the modulation of inflammatory factors produced during the various non-thyroidal diseases. Thirdly, the lack of data regarding serum total T3 levels and cognitive function within normal thyroid function, with low levels of FT3 associated with the risk of cognitive impairment. Additionally, TSH and FT3 levels also impacted the cognitive domain of immediate memory. As these findings are derived from cross-sectional data, further confirmation is needed from a longitudinal exploration of impact of FT3 supplementation on the cognitive function of euthyroid older adults.In conclusion, euthyroidism is mostly neglected by researchers. However, this state is also essential in the community, particularly among older adults with cognitive decline. Our findings from older adults in the Chinese community suggested an independent relationship between serum FTThis is a cross-sectional analysis of the baseline survey data obtained from the cohort study on the health status of rural older adults from the Guizhou province in China (HSRO). The data were obtained using multistage cluster sampling. A total of 12 villages in the province were selected, and the baseline survey was conducted from July to August 2019. The inclusion criteria included community-dwelling volunteers aged ≥ 60 years who were long-term residents of the area (at least > 6 months). A total of 1795 older adults were enrolled in this study. Among them, 560 individuals were excluded for the following reasons: (i) did not cooperate in the blood sample collection, (ii) did not have normal communication skills, (iii) had been diagnosed with severe mental illness by the medical center (including severe depression and anxiety), and (iv) had taken drugs that affected thyroid function, such as levothyroxine and thyroxine. Thus, 1235 community-dwelling older adults were included in the thyroid function assessment analysis, of whom 957 were euthyroid individuals. The study was approved by the Ethics Committee of Guizhou Medical University, and all the participants signed informed consent.4), and free triiodothyronine (FT3) were determined using the Roche Cobase601 automated chemiluminescent immunoassays . The reference ranges provided by the manufacturer were as follows: FT3, 3.10 ~ 6.80 pmol/L; FT4, 12.00 ~ 22.00 pmol/L; and TSH, 0.27 ~ 4.20 mIU/L.Blood samples were collected the morning after an overnight fast. Serum levels of TSH, free thyroxine ; overt hypothyroidism (TSH > 4.20 mIU/L and FT4 < 12.00 pmol/L); subclinical hypothyroidism (TSH > 4.20mIU/L and FT4 = 12.00 ~ 22.00 pmol/L); subclinical hyperthyroidism  and overt hyperthyroidism 32.Participants were categorized based on TSH, FT33. MMSE results can quickly reflect a global cognition in clinical, research, and community settings. The test comprises 11 items divided into five domains: orientation, immediate memory, attention, language, and delayed recall. Trained investigators recorded the participants’ responses while observing their behavior and then summed the scores assigned to each question. The 30-point questionnaire contains a total of 30 test questions. For each correct response, the participants would score 1 point; thus, each participant could obtain a total score of 0 to 30 points. Cognitive impairment was defined as MMSE score of ≤ 17 for illiterate participants,  ≤ 20 for those with primary school education and below, and ≤ 24 for those with junior high school education and above34.The mini-mental state examination (MMSE) score was used to assess the cognitive function of participantsIn this study, we examined several demographic characteristics of the participants, including gender, marital status , and education , age . Based on their smoking status, the participants were divided into two categories: currently smoking  or not smoking (including quitting smoking (defined as quitting for > 6 months) and never smoking). The drinking category was divided into 2 categories: currently drinking (defined as drinking on an average of ≥ 1 days per week in the past year) or not drinking . Hypertension was assessed through self-report; participants needed to answer the question, “Have you ever been told by a doctor, nurse, or another health professional that you have hypertension?” Responses were grouped into two categories: Yes and no.35.Utilizing the Patient Health Questionnaire-2 (PHQ-2), the evaluation of depressive symptoms was conducted. Comprising two items, this questionnaire probes into the frequency of depressive mood and feelings of loss over the preceding two weeks. Response options, including "not at all", "a few days", "more than half of the days", and "almost every day", are associated with scores of 0, 1, 2, and 3, respectively. Consequently, PHQ-2 scores span from 0 to 6, with a score of 3 or more indicating the potential presence of a depressive disorder36.For the assessment of anxiety symptoms, the Generalized Anxiety Disorder 2-Item Scale (GAD-2) is employed as a concise and user-friendly tool designed for the initial screening of generalized anxiety disorder. This scale consists of two items that investigate the frequency of anxiety over the past two weeks. Response options, ranging from "not at all" to "almost every day", are correspondingly assigned scores of 0, 1, 2, and 3. Consequently, GAD-2 scores range from 0 to 6, and a score of 3 or higher suggests the potential presence of an underlying anxiety disorderP < 0.05.All statistical analyses were performed using SPSS for Windows version 22.0 . MMSE scores and, THs were analyzed by quartile grouping in order to observe cognitive differences. Spearman’s correlation coefficients were calculated to estimate the association of MMSE scores with thyroid hormones. Bootstrapping was used to obtain 95% confidence intervals (CIs). Multiple linear regression and binary logistic regression analysis were used to assess the relationship between THs and cognitive function (domains) after adjustment for age, gender, marriage, and other relevant variables . The difference between all subjects and euthyroidism was assessed and shown in the appendix. All statistical tests were 2-sided and the difference were considered significant at Written informed consent was obtained from each participant before any study procedure was initiated, and the collection of data on human subjects was approved by the medical ethics committee of Guizhou Medical University . All methods in this study were performed in accordance with the guidelines of the Declaration of Helsinki.Supplementary Tables.


Candida albicans isolates and anidulafungin showed a major discrepancy (MD) (2.60%), and no other MD or very MD agents were found. Therefore, Droplet 48 can be considered as an optional method that is more automated and can obtain results and interpretations faster than previous methods. However, the optimization of the detection performance of posaconazole and voriconazole and promotion of Droplet 48 in clinical microbiology laboratories still require further research involving more clinical isolates in the future.Fungal pathogens are a major threat to public health, as they are becoming increasingly common and resistant to treatment, with only four classes of antifungal medicines currently available and few candidates in the clinical development pipeline. Most fungal pathogens lack rapid and sensitive diagnostic techniques, and those that exist are not widely available or affordable. In this study, we introduce a novel automated antifungal susceptibility testing system, Droplet 48, which detects the fluorescence of microdilution wells in real time and fits growth characteristics using fluorescence intensity over time. We concluded that all reportable ranges of Droplet 48 were appropriate for clinical fungal isolates in China. Reproducibility within ±2 two-fold dilutions was 100%. Considering the Sensititre YeastOne Colorimetric Broth method as a comparator method, eight antifungal agents  showed an essential agreement of >90%, except for posaconazole (86.62%). Category agreement of four antifungal agents  was >90%, except for voriconazole (87.93% agreement). Two  Key inthttp://www.eucast.org/ast_of_fungi/). The BMD methods, as gold standards for AFST, are labor-intensive during microdilution plate preparation and have expensive antifungal powders; therefore, it is impractical for routine use in clinical microbiology laboratories  that correspond to approximately 20 bacterial cells, or a single fungal cell in the detection volume , uses a unique fluorescence detection technology to feed back the growth of pathogens through continuous detection of changes in fluorescence intensity of the panel; it reports the results in real time. It also avoids the subjectivity of interpreting results by reading MICs manually, and is simple to apply in clinical microbiology laboratories.Candida krusei as an example, the D48 detector fitted the acquired fluorescence signal to the growth curve and determined the MIC using various algorithms  is a water-soluble dye that can be transformed into a fluorescent colorimetric indicator (pink) using metabolically active cells. When resazurin concentration was sufficient, the fluorescence intensity produced by resazurin was proportional to the number of viable cells . Based oCandida albicans entered the logarithmic growth phase at approximately 15 h, while Exophiala dermatitidis reaches it at approximately 45 h . To fully validate the performance of D48, we included 10 strains of Trichosporon asahii isolated from patients with bloodstream infections, 22 strains of Exophiala dermatitidis causing oral mucosal infections, and 11 randomly selected isolates from patients with rare bloodstream infections.A total of 144 clinical fungal strains were randomly analyzed from the Peking Union Medical College Hospital and Peking University School and Hospital of Stomatology. Species of these strains were commonly found in patients with systemic infections, including 16 The CHROMagar Candida chromogenic agar medium was used for initial identification. Species identification was further confirmed using matrix-assisted laser desorption ionization-time of flight mass spectrometry and sequencing of the nuclear ribosomal internal transcribed spacer region and large subunit of the 28S ribosomal DNA gene (D1/D2).2.23 CFU/mL and 0.5–2.5 × 103 CFU/mL, respectively  was performed, according to the manufacturer’s instructions. To prepare the inoculum, all the strains were subcultured onto sabouraud dextrose agar or potato dextrose agar at 35°C and subcultured again to ensure purity and viability  and epidemiology cut-off values (ECVs) in the 2022 CLSI files [M57S 4th Edition .3Saccharomyces cerevisiae strain could not produce the desired results using the D48 method, and one Rhodotorula mucilaginosa strain could not obtain the desired results using the SYO method. Therefore, 142 samples were included in the statistical analyses.The MICs for the quality control strains were within the recommended ranges of the CLSI for both assays. This study included 144 strains. Owing to inadequate growth, one The reportable range of D48 fully covered the CBs, ECVs, and quality control ranges of common clinically pathogenic fungi. Combined with microfluidics and other technologies to increase the concentration gradient of more drugs, D48 realizes simultaneous detection of 96 different drug concentrations. Except for 5-flucytosine and voriconazole, there was a slight difference in the reportable ranges between the two methods; however, the ranges were appropriate for clinical fungal isolates from China Table 1.Candida albicans and anidulafungin showed MD (2.60%); no other MD or VMD agents were found. Voriconazole (12.07%), fluconazole (7.69%), caspofungin (6.41%), anidulafungin (3.85%), and micafungin (1.28%) were the agents with CBs that all showed mD. Categorical agreement analyses were performed for strains and agents with CBs only, and we used the Cohen’s kappa coefficient for further analysis of all the strains and agents with CBs or ECVs. We noticed an almost perfect degree of detection agreement between the D48 and SYO for fluconazole, voriconazole, itraconazole, caspofungin, micafungin, anidulafungin, and 5-flucytosine (Cohen’s kappa coefficient ≥0.81). Posaconazole (Cohen’s kappa coefficient: 0.06) showed slight agreement, whereas amphotericin B (Cohen’s kappa coefficient: 0.47) showed moderate agreement  and D48 Table 1.Trichosporon mucoides and caspofungin/posaconazole, Candida norvegensis and anidulafungin, Saccharomyces cerevisiae and amphotericin B/fluconazole was more significant than two two-fold dilutions with SYO. The remaining samples (EA) were 100%. Although we could not interpret the consistency of the two detection methods due to the small number of strains, the MIC values of the tested strains were relatively consistent, which could provide a therapeutic option for patients with clinically rare fungal infections.In recent years, there has been growing interest in cases of uncommon and unknown “superfungal” diseases. In our study, 11 uncommon clinical fungal isolates from the bloodstream were included, and 4A novel automated antimicrobial susceptibility testing system, D48 , based on the growth characteristics of pathogenic fungi, was evaluated in this study. It is real-time, quick, affordable, and simple to promote in clinical pathogenic microbiology laboratories, and avoids the subjectivity of manually reading MICs. The Fungi and Mycosis Research Center of Peking University compared the detection results of D48 and CLSI BMD methods when applied to the China Medical Device Product Registration Certificate (No.20192220368 and No.20192400349), and the EA of nine antifungal drugs were all >90%, which is consistent. Therefore, this study did not compare D48 with the BMD reference method.According to the CLSI criteria for the verification of commercial antimicrobial susceptibility testing systems, SYO approved by the U.S. FDA was chosen as the comparator method. Importantly, the CLSI M52 document stipulates that when a clinical laboratory desires to implement a new AFST system, it should be compared with the commercial method that the laboratory is already using and meets the criteria.The D48 should be verified for specific agent/fungus combinations tested when 1) CA and EA (if reporting MICs) are ≥90% compared to that in the current system; 2) VMD rate was <3% of total resistant isolates; and 3) MD rate was <3% for all susceptible isolates . AccordiCandida albicans, one Cryptococcus neoformans, and one Trichosporon asahii had inconsistent amphotericin B ECVs between the two methods, thereby leading to this discrepancy. Because the EA of the two methods was better than 90% for amphotericin B, we believe that the D48 has excellent performance in detecting amphotericin B.The Cohen’s kappa coefficient for amphotericin B was 0.47, while the EA rate was 95.07%. The Cohen’s kappa coefficient was calculated for strains with ECVs values, which reduced the sample data size compared with the EA included in all samples for calculation. Additionally, one Candida glabrata and caspofungin, followed by 75% for Candida albiacans and voriconazole, 81.25% for Candida albicans and anidulafungin, 84.62% for Candida parapsilosis and fluconazole, and 87.50% for Candida krusei and voriconazole/caspofungin. The CA of the other strains and agents were >90%. Except for Candida albicans and anidulafungin whose MD was 12.5%, none of the isolates showed VMD or MD. Candida glabrata and caspofungin were associated with up to 30% mD, followed by Candida albicans and voriconazole (25.0%), Candida parapsilosis and fluconazole (15.38%), and Candida krusei and voriconazole (12.50%). The test range, MIC50, MIC90, and agreement rate between D48 and SYO for each species are shown in The agreement between the two methods has also been evaluated in different species. The lowest CA rate (70%) was found for Candida albicans and Candida tropicalis, which appeared as slight color changes and persisted in microdilution wells with concentrations greater than the MIC value .All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.


This study aimed to evaluate the feasibility and limitations of thoracoscopic segmentectomy of the basal segment (S10).Clinical data of 15 children with congenital lung malformations (CLM) who underwent thoracoscopic segmentectomy of S10 via the inferior pulmonary ligament approach from January to October 2022 were retrospectively analyzed. The demographics, clinical presentation, intraoperative time, blood loss, postoperative events, and follow-up duration were assessed.There were 15 patients in this group . Age ranges from 4.3 to 96.0 months . Fourteen patients underwent S10 segmentectomy, with one undergoing right S10 segmentectomy and right S6 partial wedge resection. The surgical time was 57–125 min , intraoperative bleeding volume , postoperative drainage tube indwelling , and postoperative hospitalization time . No intraoperative conversions, surgical mortalities, or major complications were observed among these patients. Subcutaneous emphysema appeared in three patients; however, it disappeared following conservative observation without pneumothorax or bronchopleural fistula occurrence.Thoracoscopic segmentectomy of S10 via the inferior pulmonary ligament approach is technically feasible for treating CLM; however, this surgical approach may have certain limitations for CLM with large cysts. Congenital lung malformations (CLMs) are characterized by airway dysplasia, lung parenchyma, and pulmonary vessels. Congenital pulmonary airway malformations (CPAM) and intra-lobar pulmonary sequestration (ILS) are the most common types . CurrentThe Ethics Committee of Jinan Children’s Hospital  approved this study, and it followed the principles of the Declaration of Helsinki. The parent of each child provided written informed consent.In our hospital, 15 children with CLM underwent thoracoscopic S10 resection through the inferior pulmonary ligament approach between January and October 2022. All preoperative patients underwent enhanced chest CT to confirm the diagnosis, determine the abnormal supply of blood vessels , and design a surgical plan was performed to achieve single-lung ventilation. An artificial pneumothorax was established at a pressure of 4–8 mmHg (1 mmHg = 0.133 kPa) and a flow rate of 1–2 L/min. A three-hole method was used to perform surgery in the side-lying position. The observation hole was located at the 7th intercostal line of the subscapular angle. The two operating holes were located at the 8th intercostal line of the axillary midline and the 8th intercostal line between the vertical spinal muscle line and the subscapular angle line. The chest cavity was accessed, and the inferior pulmonary ligament was severed with an electric hook to expose the lower pulmonary vein and totally free the lower pulmonary vein tributaries (V6) and common basal vein (CBV). Next, dissociate V10 along the CBV, detach V10, and completely dissociate V9 from its deep surface. Next, locate and totally separate A10 and B10 between V6 and V9 . Table There were 15 patients in this group . Age ranges from 4.3 to 96.0 months . Weight ranged from 7.0 to 27.0 kg . Thirteen cases of CLM were diagnosed using prenatal ultrasound and chest CT examination within 3 months after birth, including seven cases of CPAM and six cases of ILS. Owing to recurrent respiratory tract infections, two patients were diagnosed using chest CT, including one case of CPAM and one case of ILS. Lesions in six cases were located on the right S10, whereas those on the left S10 were found in nine. The lesion diameters of the lesions range from 2.8 to 5.9 cm . Six children underwent surgery following rehabilitation for recurring clinical respiratory symptoms (cough in four cases and wheezing in two). Nine were asymptomatic, but their parents requested active surgical treatment , no patients presented with hemoptysis, and no residual lesions were observed on chest CT. In addition, symptoms either completely resolved or significantly improved in all symptomatic patients, and no complications were detected throughout the follow-up period.The incidence of CLM is 1–4.2 per 10,000 . The CLMOwing to the fact that CLM is a benign lesion, segmentectomy is a good choice for patients whose lesions are limited to the lung segment if the technical level is mature. Lung segments were divided into simple or complex segments. Simple segmental resection, similar to lobectomy, is a relatively simple and widely performed surgery. Complex lung segment resection is challenging because of the need to manage multiple segment planes. S10 Surgery is more challenging since it is far from the hilum of the lungs and adjacent to three or four lung segments , 13. SegChildren have a smaller thoracic space than adults do, and an artificial pneumothorax is more likely to affect hemodynamics significantly. Therefore, establishing a suitable operating space is critical to a successful surgery. By combining an artificial pneumothorax with selective single-lung ventilation, we established a surgical operating space. During surgery, we found that the inferior pulmonary ligament approach made it easier to construct an operating area. Only one-lung ventilation in the lower lobe is required to obtain sufficient operating space because there is no need to consider the impact of the upper and middle lobes on the operating space after establishing an artificial pneumothorax and completing diaphragm compression. This significantly reduces the technical requirements for anesthesiologists and makes surgery easier. It is necessary to break the cysts to gain a larger space for patients with CLMs and large cysts . CLM difOur center has achieved good short-term clinical outcomes in treating CLM using thoracoscopic segmentectomy of S10 via the inferior pulmonary ligament approach. However, the sample size of this study was small, and it was a single-center, retrospective study. Therefore, it will be necessary in later stages to further verify the feasibility and limitations of this surgical method in treating patients with CLM using large-sample, prospective, and multicenter studies. In this study, we found that there are challenges in determining the inter-segment plane and performing precise segmentectomy of S10 using only the modified “dilation and collapse method” for CLM with large cysts. Therefore, other surgical methods are more suitable for these patients. Some researchers have recently proposed surgical methods, such as “modified wedge resection” and “anatomical lesion resection.” Theoretically, the core of these surgical methods is to perform precise resection of the lesion via inter-segmental veins as the internal boundary and the natural boundary between the lesion and normal tissue as the external boundary, rather than being limited to precise segmentectomy , 24. TheThoracoscopic segmentectomy of S10 via the inferior pulmonary ligament approach is technically feasible for treating patients with CLM; however, this surgical method may have certain limitations for CLM with large cysts.


When AUC0.25h and AUC0.5h were considered, slower rates of absorption were also seen for lozenges and spray gel. The differences correlated with the reported time of onset of action, which is faster for the spray solution (20 min) compared to lozenges (26 min) and granules (30 min). These pAUCs provide useful data that allow for the discrimination between formulations. Moreover, the pAUC values represent <5% of the total AUC, suggesting that the early onset of pain relief is a response to immediate local absorption at the site of action rather than a systemic effect.This study aimed to link pharmacokinetic (PK) data from different flurbiprofen preparations for the treatment of sore throat with published data to elucidate whether early efficacy is due to the local action of flurbiprofen or a systemic effect after absorption of the swallowed drug. Three comparative bioavailability studies conducted in healthy subjects provided data from flurbiprofen 8.75 mg formulations, including spray solution, spray gel, lozenges, and granules. A parallel interstudy comparison was made of PK parameters, including partial AUCs (pAUCs), using an ANOVA model with the calculation of 90% confidence intervals (CI) for the differences between least squares (LS) means for each of the test groups versus the respective reference groups. All three studies showed bioequivalence for the respective product comparisons. The interstudy comparison showed a slower rate of absorption for granules compared to spray solution (reference) based on T The main objective of developing locally applied products, including non-steroidal anti-inflammatory drugs (NSAIDs), is to ensure that they are delivered locally and exert their effect only at the locally affected site, with any systemic effects being considered undesirable ,2. The sFor example, in a study in healthy subjects that compared topical diclofenac sodium 2% solution twice daily and diclofenac sodium 1.5% solution four times daily to oral diclofenac sodium 75 mg tablets twice daily, the 2% and 1.5% topical solutions were bioequivalent with respect to AUC at steady state, with approximately 93% lower systemic exposure compared to oral administration . TopicalThe efficacy of locally applied, locally acting NSAID products is a consequence of higher concentrations achieved in the target tissues when compared to those seen in the systemic circulation. Topical diclofenac or flurbiprofen, for example, are associated with better penetration ,12 and pSimilar data have been reported for flurbiprofen. For example, in patients administered flurbiprofen 40 mg perorally or topically 16 h and 2 h prior to knee surgery, significantly higher concentrations of the active drug were found in the fat, tendon, muscle, and periosteal tissues in the topical application group compared to those who received tablets .Although the efficacy and safety of locally applied NSAIDs are well established in a variety of indications, the role of local versus systemic concentrations in the pharmacological effects remains incompletely understood. For flurbiprofen 8.75 mg lozenges and sprays used in the treatment of oral inflammatory conditions, in vitro data show that the active drug reached all layers of the human cadaveric pharynx mucosal tissue, including the underlying lamina propria, which contains blood vessels and nerve fibres that contribute to pain during pharyngitis ,13. ThisIn this study, we compared the PK parameter (pAUC) of different locally applied flurbiprofen preparations with the efficacy parameter  from published studies of the treatment of sore throat. Such a comparison contributes to the elucidation of whether initial efficacy is due to local absorption in the throat rather than systemic absorption of swallowed flurbiprofen from the GI tract.2. The studies were conducted in accordance with the Declaration of Helsinki and Good Clinical Practice recommendations. Approvals were obtained from an ethics committee in line with local regulations, and written informed consent was obtained from each participant. The studies were registered on the European Union Drug Regulating Authorities Clinical Trials Database (EudraCT).Three randomised, open-label, crossover design PK studies were conducted in healthy male and female subjects following standard inclusion/exclusion criteria for bioavailability and bioequivalence studies ,18, inclStudy EudraCT 2018-003175-36 included 16 subjects and compared two flurbiprofen viscous spray gel formulations to a reference spray solution (non-viscous). The treatment regimen for all products was a single therapeutic dose of 3 sprays  delivered to the back of the throat.The study EudraCT 2011-003332-31 compared two spray solutions  to a flurbiprofen 8.75 mg lozenge formulation (Treatment A) in 33 subjects.Finally, the randomised study EudraCT 2008-005177-34 consisted of 16 subjects using flurbiprofen granules or a lozenge formulation, both at a strength of 8.75 mg, crossing over to the alternative treatment in the second period.max [max), area under plasma concentration curve from administration to last quantifiable concentration at time t (AUC0−t), time to maximum observed concentration (Tmax), AUC extrapolated to infinity (AUC0−inf), elimination rate constant (Kel), and elimination half-life (T½). An Analysis of Variance (ANOVA) model (separate models for each product) using Excel was fitted to naturally log-transformed (ln) AUC0−t, Cmax, and AUC0−inf with fixed terms for treatment, period, sequence, and subject nested within sequence, and 90% CI for the differences between LS means for each of the test groups versus the chosen reference group were calculated for each of the individual PK studies.A total of 12, 18, and 15 blood samples, respectively, were drawn over 720 min from each subject at each study period in the first, second, and third studies; all studies included sampling at least every 5 min for the first 15 min , along with further sampling at 30 min. The sampling time points were based on standard requirements of adequate sampling prior to and around the Cmax ). The plmax . The folmax ): maximuPlasma concentration data from all three PK studies was combined into one database. Partial AUCs (pAUCs) over the first two hours after administration of flurbiprofen products were calculated using the linear trapezoidal rule as an additional metric to reflect the rate of absorption ,23. The The results of the study EudraCT 2018-003175-36 in 16 subjects are presented in 0−t ranging from 3930 ng∗h/mL (spray gel B) to 4225 ng∗h/mL (marketed spray solution), denoting a 7% difference between the extremes. As a measure of rate of absorption, the geometric means for Cmax ranged from 922 to 1040 ng/mL (highest for the marketed spray), with a 12% difference between the extremes. Both spray gel formulations were bioequivalent to the reference spray solution product, with 90% CI for AUC and Cmax falling within the standard acceptance range of 80–125%, apart from the lower CI for Cmax for the second spray gel formulation (B), which was marginally below 80%. No statistically significant differences were observed in secondary PK parameters  when the new spray gel formulations were compared with marketed spray solutions.The PK parameters were similar for extent of absorption, with geometric means for AUCThe results of study EudraCT 2011-003332-31 in 33 subjects (presented in 0−t geometric means ranging from 5544 to 5682 ng∗h/mL) and by less than 2% for rate of absorption (Cmax geometric means ranging from 1553 to 1580 ng/mL). The 90% CIs for the ratios of the geometric means for Cmax and AUC0−t fell within the standard acceptance range of 80–125%, confirming the bioequivalence of the spray solutions compared to the lozenges. There was a statistically significant difference between Tmax values , with plasma concentrations peaking earlier for the spray solutions (median 0.50 h) than for the lozenges (median 0.83 h), a difference of well over 20%.The spray solutions (Treatments B and D) and lozenges (Treatment A) differed by less than 3% for extent of absorption , as shown in 0−t) was similar for granules and lozenges, with geometric means (5932 and 6251 ng∗h/mL) differing by approximately 5%. For Cmax as a measure of rate of absorption, the respective geometric means were 1413 and 1620 ng/mL, just less than 13% lower for granules compared to lozenges. The formulations were bioequivalent, with 90% CIs for the ratios of geometric means for Cmax and AUC0−t falling within the standard acceptance range of 80–125%. There was a statistically significant difference between formulations for peak plasma concentrations ; although median Tmax values for the lozenge formulation (0.75 h) differed from granules (0.88 h) by less than 20%, the respective ranges differed markedly (0.50 to 1.00 h for lozenges and 0.25 to 2.00 h for granules), and the difference between arithmetic mean Tmax values was ~25 min.The extent of absorption (AUC0−t) but suggested possible differences for rate of absorption as measured by Cmax, with a slower rise in plasma levels for granules when compared to spray solution  with respect to extent of absorption . A similar pattern was also observed for the comparison between one of the spray gel formulations (A) and the spray solution, with a significant difference detected for AUC0.25h and a trend for a difference for AUC0.5h . The behaviour of the spray gel formulation (B) was not statistically different from the spray solution for any of the pAUCs.The pAUCs at these earlier time points more clearly indicate possible differences between the formulations. As shown in In order to elucidate the possible links between therapeutic effect and early exposure, the pAUCs for 0–15 min and 0–30 min were compared and correlated with the previously published onset of action data, specifically the time to clinically meaningful pain relief for the different formulations .Linear correlation coefficient values were close to 1, strongly supporting a link between the extent of absorption of flurbiprofen in the first 15–30 min and the timing of onset of action  as reported in previously published therapeutic trials ,25.Currently, flurbiprofen is the only low-dose NSAID ,15 for o0−t values. Moreover, the rate of absorption as expressed by Cmax values is also similar, with a 90% CI within or close to the standard bioequivalence range of 80–125%, albeit with slightly slower absorption for granules when compared to lozenges . In contrast, it is known that different topical pharmaceutical forms of flurbiprofen , lozenges, and granules, all exhibit a very similar extent of absorption as shown by similar  AUC. TH0918 ), lozeng. TH0918 ,15,16,250−t and Cmax, do not directly and strongly reflect comparable efficacy [Given the differences in time to onset of clinically meaningful pain relief for the different formulations that met bioequivalence criteria (90% CI within 80–125%) , conventefficacy  for thesefficacy , createdmax and lower Cmax for the granule formulation described above. In contrast, for lozenges vs. spray solution, the 90% confidence intervals for AUC1h and AUC2h fell within standard bioequivalence limits, again suggesting no clinically relevant difference between formulations. This finding is consistent with clinical observations made in therapeutic non-inferiority studies, which demonstrated similar efficacy for these respective formulations at 1 h [1h and AUC2h within the standard bioequivalence limits and a lower limit of the 90% CI slightly below the lower acceptance range when compared to spray solution  and had similar AUC0.5h values , in contrast to later values (AUC1h and AUC2h), distinguish between formulations  that have a similar overall extent of absorption and similar peak plasma concentrations but have been shown to have different times to onset of action in therapeutic trials. The fact that these AUC0.25h and AUC0.5h values represent no more than 1 to 5% of the total AUC suggests that the early onset of pain relief is a response to immediate local absorption at the site of action rather than a systemic effect. This, in turn, would support the use of early pAUCs for clinical effect correlation of locally applied, locally acting products and for head-to-head comparisons, establishing the bioequivalence of different formulations.Finally, to better understand the potential link between these earlier pAUCs and the onset of action, a correlation was performed between the absolute values of AUCmax and AUC(0−t) may be insufficient to adequately assess bioequivalence between two products, in particular when early onset of action is clinically relevant. In these cases, partial AUC may be applied, most typically from the time of drug administration until a predetermined time point that is related to a clinically relevant pharmacodynamic (PD) measure.Partial AUCs are already currently used by some agencies to better characterise the PK profiles of certain products. For instance, the FDA recommends the use of pAUC as an exposure measure in a number of product-specific bioequivalence guidelines, mainly for certain modified-release (MR) products in which the different phases of release correspond to a clinical effect . EuropeaThe data presented above show that for certain products, such as locally applied, locally acting NSAIDs for use in the oral cavity and throat, pAUCs provide useful data that allow discrimination between formulations. Moreover, these data may be more clinically relevant than those gained through, e.g., charcoal blocks.It is acknowledged that the exact PK/PD profile of locally applied flurbiprofen has not been reliably established as the presented work is based on parallel study comparisons and retrospective data analyses. The link between PK, local concentrations, and onset of action should be further elucidated in a single study in order to confirm the potential use of early pAUCs as a surrogate for comparison of early pain relief in products of this type.While the ultimate proof of similarity across different formulations of locally applied and locally acting drugs in the mouth and throat usually requires therapeutic studies, the comparison of pAUCs provides a promising alternative. The possibility of using PK data, in particular pAUCs, with associated PD correlations to assess the therapeutic equivalence of two formulations containing similar doses of active for local application and local activity in the mouth and throat should be reflected in the equivalence guidelines.The information on gel spray has been submitted for a patent, and further details on this patented formulation are not available for this manuscript.


The kinematic study showed that the affected limb group had smaller hip adduction angle (p = 0.002) and increased internal rotation (p = 0.006) with reduced upward pelvic tilt (p = 0.020). Osteonecrosis was graded II, III, and IV in five, three, and one patients, respectively. Five patients had no AVN changes. The Severin classification was grade I, II, and III for six, three, and five patients, respectively. Most patients had good functional outcomes on the CHOHES, with a mean total score of 96.64 ± 5.719. Multivariate regression analysis revealed that weight, height, and femoral osteotomy were independent predictors for gait, radiological and functional outcome. Conclusion: Despite good functional scores overall, some children had poor radiological outcomes and gait abnormalities. Our results identified the risk factors for poor outcomes, and we recommend specified rehabilitative strategies for long-term management.Background: Children undergoing DDH correction surgery may experience gait abnormalities following soft tissue releases and bony procedures. The purpose of this study was to compare the residual gait changes, radiological outcomes, and functional outcomes in children who underwent DDH surgery with those in healthy controls. Methods: Inertial motion sensors were used to record the gait of 14 children with DDH and 14 healthy children. Pelvic X-ray was performed to determine the Severin classification and the presence of femoral head osteonecrosis (Bucholz–Odgen classification). For functional evaluation, the Children’s Hospital Oakland Hip Evaluation Scale (CHOHES) was used. Results: There was no difference in spatial parameters between the two groups. In terms of temporal parameters, the DDH-affected limbs had a shorter stance phase ( Developmental dysplasia of the hip (DDH) is a spectrum of abnormal development that results in acetabular dysplasia, hip subluxation, and possible dislocation due to capsular laxity and other mechanical factors. It is known to be the most common orthopedic disorder in newborns, with an incidence of 1:100 for dysplasia and 1:1000 for hip dislocation ,3,4,5. AIn this study, we hypothesized that the surgically corrected DDH patients would show gait deviations after hip surgery. Information gathered from this study will help researchers understand and correlate gait deviations to the surgical procedure and the radiological and functional outcomes. Researchers will be able to improve their knowledge regarding the postoperative surgical outcomes of DDH and the impact on gait pattern. This study is a prospective case-control study conducted at a tertiary hospital for the duration of 18 months. A total of 14 DDH patients , who were surgically treated, were a minimum of 2 years postsurgery , and aged between 8 and 18 years old were included in this study . The folFor gait analysis, the patients were asked to walk at their own pace in a straight line on a 10 m walkway while wearing the Xsens Awinda inertial motion sensors  . The datt-test was used to analyze numerical data with the means. Pearson correlation was performed to determine the association between the risk factors and the outcome measures. A difference was considered statistically significant at p ≤ 0.05. Statistical analysis was performed using SPSS version 26 . Descriptive data re expressed as mean ± standard deviation (SD) unless otherwise stated. ANOVA was used to analyze categorical data, while the The characteristics of the participants are described in p = 0.002) for the control group; and the maximum hip rotation  had a deviation mean value of 4.96 ± 5.48 for the affected group compared with 0.43 ± 3.89 (p = 0.006) for the control group. The DDH-affected limb appeared to have significantly less hip adduction when walking compared with the limbs of the control group, and even with the unaffected limb (0.045). In terms of the compensatory effect of the DDH-affected limb on the unaffected limb, no significant kinematic parameters could be observed. In terms of knee and ankle flexion and extension, no significant difference could be seen for the deviation mean between the affected and control groups. Regarding the pelvic motion parameters, it was seen that the affected limb group had less upward hiking compared with the limbs of the control group in the coronal plane (p = 0.020).In terms of spatial parameters such as speed, cadence, step width, step length, and stride length, no difference between the DDH group and the control group was observed. For temporal parameters, there was a significant increase in swing phase duration, swing phase per gait cycle, single support phase duration, single support phase per gait cycle, midstance duration, and midstance per gait cycle in the DDH group compared with in the control . SimultaWith regard to the radiological outcomes of the 14 patients, 5 (No AVN), 5 (grade II), 3 (grade III), and 1 (grade IV) patients were classified according to the Bucholz and Olden classification. In contrast, six (grade I), three (grade II), and five (grade III) patients were classified according to the Severin classification.Measurements of intra- and interobserver variability obtained an intraclass correlation coefficient (ICC) value of 0.96 and 0.93, respectively, with a 95% confidence interval. This indicated excellent reliability because the ICC values for both observers are greater than 0.75.Utilizing the CHOHES functional assessment tool, most of the patients exhibited good functional outcomes with a mean of 96.64 ± 5.719 in total score. The mean scores for each of the domains, which consist of pain, function, and physical examination, were 39.29, 30.57, and 26.79, respectively. For the range of motion outcomes obtained from the physical examination among the 14 children with DDH, the results were as follows: (a) hip internal rotation, 0 (<16°), 1 (16–29°), 1 (30–39°), and 12 (>39°); (b) hip external rotation 0 (<16°), 1 (16–29°), 2 (30–39°), and 11 (>39°); (c) hip flexion 0 (<90°), 2 (90–100°), 5 (101–114°), and 7 (>114°); and (d) hip abduction 0 (<20°), 0 (21–29°), 0 (30–39°), and 14 (>39°), respectively.The association of each patient’s characteristics with the measured outcome is depicted in The multiple linear regression analysis to control the confounding effects of the variables is shown in The results of the current study demonstrated the existence of gait deviations in the surgically corrected DDH patients group compared with the control group. Furthermore, the comparable demographic characteristics between the case and control groups justified the use of this population as a baseline for healthy gait. Although comparing the postoperative gait of DDH children with healthy children might be less than ideal, a comparison of the pre- and postoperative gait in the same patient was not possible as the patients had their surgery before, or at, walking age . NeverthAs for the spatial parameters in the gait analysis , no significant difference was seen between the DDH and the control groups. A similar outcome was also reported by Ömeroğlu et al., where the mean velocity and step-length values of the soft tissue release surgery-affected and unaffected sides were similar to those of the healthy control group . In anotIn terms of the kinematic parameters, the evaluation of the range of motion of the pelvis, hip, knee, and ankle in the coronal, sagittal, and axial plane revealed an increase in hip internal rotation during walking in the affected hip when compared with those of the control group. This could be due to DDH patients’ abnormal proximal femoral morphology in the first place. DDH patients have an abnormal rotational profile at the proximal femur, which may cause an increase in internal rotation in the affected hip in addition to a dysplastic acetabulum ,30. By uWhen compared with the control group, the affected hip showed reduced adduction during walking. This could be attributed to the weakness of the hip adductor of the affected hip, because adductor tenotomy was performed during surgery to help reduce the hip and increase the safe zone of Ramsey for postoperative application of the hip spica. Interventions to strengthen hip adductor muscle strength and flexibility, particularly static stretching, have demonstrated restoration of hip adduction in 40 patients with limited hip adduction . Thus, iA weak hip abductor, which is common in hip pathology, may also cause an increase in hip internal rotation. Weakness in the hip abductor could explain the reduction in pelvic upward tilt in the coronal plane observed in this study. Compared with the normal control group, the DDH-affected hip showed a statistically significant reduction in upward pelvic tilt. Similar findings were described in DDH patients in their adulthood when compared with the control population following triple innominate osteotomy or total hip arthroplasty procedures ,33. ThusRegarding gait temporal parameters, the DDH-affected hip had a shorter stance duration and percentage, as well as a longer swing phase duration and percentage, when compared with the limbs of the normal group. This finding could be attributed to patients offloading their pathological hip earlier in order to avoid causing discomfort to the affected hip (1). Chang et al. obtained a similar result, with a significantly shorter terminal double limb stance (DLS) in the affected limb after Pemberton osteotomy. Similarly, Nie et al. reported shorter DLS and single limb stance (SLS) among DDH patients who underwent unilateral THA surgery compared with the normal healthy individual, although the differences were not significant between the two groups . When coThe strength of the current study relies on the multivariate regression analysis of risk factors influencing the outcome of DDH correction. This allows the identification of independent predictors of the outcome of DDH correction after adjusting for other confounding factors. In terms of gait variables, it was revealed that increasing weight independently predicted the increase in downward and backward pelvis tilt. Furthermore, increasing weight was also found to be an independent predictor of an increase in gait cycle duration (s), while greater height and femoral osteotomy were found to reduce the gait cycle duration. Finally, increasing age at surgery resulted in a decrease in the stance phase duration (s). The interactions between all the aforementioned risk factors explained the consequence of the late intervention of DDH.After adjusting for other confounding factors that may influence the Severin classification outcome , femoral or combination  osteotomy and open reduction surgery were found to be independent predictors of lower odds of having severe deformity of the hip joint. However, this finding could have been influenced by the single sample available for each surgery type. The relationship between the radiological outcome and the type of surgery varies in the literature. Better radiological outcomes have been associated with open reduction surgery alone without the additional bony procedure, because children requiring more complex surgery may already have poor baseline radiological outcomes compared with those who underwent open reduction alone . Neverthp < 0.05): the mean CHOHES score was recorded as 95.7 (84–100) for patients younger than four years during surgery compared with 92.5 (82–100) for patients older than four years during surgery. Similarly, numerous studies have established age as a predictor of poor clinical outcomes [Moreover, increased age at surgery and weight were found to be associated with a reduced likelihood of having a high CHOHES score. In this study, when age was considered as a categorical value (being younger or older than four years during surgery), there was a significant difference (outcomes ,37,38,39outcomes . In addiIn summary, although our patients demonstrated good functional outcomes, there were significant gait abnormalities that may affect these children in the long run. However, there are some limitations in this study. First, the sample size is relatively small; hence, the actual amplitude of gait deviation in postsurgically treated DDH patients may not have been adequately collated. Moreover, this is a retrospective study analyzing the outcomes of various surgeries; thus, the outcomes are fairly heterogenous. Further research conducted in a larger population with standardization of the type of surgery performed may produce more accurate results. Ideally, a follow-up gait analysis can also be performed following rehabilitative interventions. Despite all these limitations, our findings suggest that gait analysis utilizing inertial motion sensors is able to detect changes that may not be discovered in routine physical examinations.The analysis of gait, functional, and radiological outcomes among children with DDH in the current study allowed for the identification of the risk factors associated with each outcome. The most important risk factors associated with most of the outcomes were the type of surgery, age during surgery, height, and weight. Surgically corrected DDH patients showed significant gait deviations, particularly in temporal parameters; therefore, our hypothesis as accepted. Consequently, information from the study can be utilized for rehabilitation and to aid researchers in devising strategies to improve prognosis and gait among children with DDH. Interventions aimed at strengthening the affected muscles that may affect the gait in DDH patients can be derived from the results presented. Gait analysis utilizing an inertial motion sensor is recommended to identify subtle gait changes in DDH patients.


The Arctic tundra is a relatively young and new type of biome and is especially sensitive to the impacts of global warming. However, little is known about how the Arctic flora was shaped over time. Here we investigate the origin and evolutionary dynamics of the Arctic flora by sampling 32 angiosperm clades that together encompass 3626 species. We show that dispersal into the Arctic and in situ diversification within the Arctic have similar trends through time, initiating at approximately 10–9 Ma, increasing sharply around 2.6 Ma, and peaking around 1.0–0.7 Ma. Additionally, we discover the existence of a long-term dispersal corridor between the Arctic and western North America. Our results suggest that the initiation and diversification of the Arctic flora might have been jointly driven by progressive landscape and climate changes and sea-level fluctuations since the early Late Miocene. These findings have important conservation implications given rapidly changing climate conditions in the Arctic. The Arctic tundra is a relatively young biome. Here, the authors sample 32 angiosperm clades encompassing 3600+ species and find that both long-term dispersal and in situ speciation may have contributed to Arctic flora assembly, in association with landscape, climate and sea-level changes since the early Late Miocene. This region has been warming at rates 3–4 times the global average over the last 50 years5. The Arctic tundra, to the north of the natural tree line, is especially sensitive to the impacts of global warming and is of protection concern7. This biome harbors distinctive biotas that can tolerate harsh environmental conditions associated with a short growing season, low average annual temperature, and extreme seasonality9. Altogether, about 2130 angiosperm species and subspecies occur in the Arctic and 84 species are endemic to this region , and then by dense coniferous forest in the cool intervals of the Eocene16. Paleoenvironmental reconstructions support a warm and ice-free environment for the Arctic during the middle Eocene (c. 45 Ma)17. Since the Eocene/Oligocene transition (c. 34 Ma), and especially the middle Miocene Climatic Optimum (c. 17–14 Ma), global climate deteriorated drastically18 and latitudinal temperature gradients increased sharply19. Macrofossil evidence suggests that the Arctic tundra developed at the end of the Neogene or in the earliest Pleistocene (c. 3–2 Ma)22, in agreement with the time when global temperatures decreased sharply18. However, molecular phylogenetic analyses indicate that the ancestors of some Arctic lineages originated in the mid- to late Miocene25, though these evolutionary studies have mostly been limited to single taxa. To determine the actual timeframe during which modern Arctic flora began to appear, a molecular phylogenetic study of multiple clades across the angiosperm tree of life is required27.The origin and evolutionary dynamics of this unique and fragile biota, however, remain poorly understood30. However, most studies focusing on source areas of Arctic species lack divergence time estimation and/or model-based ancestral range reconstruction29. To date, we know little about the colonization dynamics of the Arctic through time. In addition to immigration, there is evidence that some in situ speciation events have contributed to the formation of the Arctic flora32. Whether both dispersal into the Arctic and Arctic in situ speciation have similar trends through time remains unknown, however, due to a lack of concerted analyses of numerous exemplar Arctic taxa.Immigration, in situ speciation, and local extinction are the mechanisms that form local communities of species. Molecular phylogenetic and phylogeographic studies suggest that Arctic plants are mainly derived from ancestral lineages that occurred in high mountains and adjacent areas to the south of the Arctic33, we also explore the potential environmental factors underlying the formation of the Arctic flora.We used a multi-taxon analysis to investigate how the Arctic flora was shaped over time. We attempt to determine the initiation age of the Arctic flora and to test whether dispersal and in situ speciation through time have similar trends. Moreover, as it is known that biotic evolution is highly correlated with geological and climatological changesTo unravel the evolutionary history of the Arctic flora, we selected 32 clades and 3626 species, of which 548 are distributed in the Arctic and 40 are restricted to the Arctic. Although it offers the most comprehensive evolutionary analysis of the Arctic tundra to date, this study only covers about 26% of Arctic angiosperm diversity. Nonetheless, we expect that the sampled taxa are representative of the Arctic flora as a whole, as our selected taxa consist of species with various habit and life history traits and belong to 10 orders and 16 families across the angiosperm tree of life , tectonic activities in Greenland and margins of Eurasia occurred , with red Fig. . Togetheiod Fig. 18. Paleoiod Fig. 36. These Ma Fig. 37.39. Accordingly, large areas of the continents experienced drying, enhanced seasonality, and a restructuring of terrestrial ecosystems38. All of these changes would have facilitated diversification of Arctic lineages. The palynological evidence shows that after 7 Ma herbs and shrubs became important elements in the Arctic flora, and vegetation openness was close to modern levels  increased sharply between 2.6 and 0.6 Ma, peaking at about 0.7 Ma (MDisE) and 1.0 Ma (MDivE) Fig. ; Table 1c.100 kyr pacing of glacial and interglacial extremes46. During the mid-Pleistocene transition (c. 1 to 0.8 Ma), large ice sheets began to display stronger quasi-100-ka cyclicity36. These extreme environmental conditions could have prevented immigrants from occupying the Arctic and might have also promoted increased extinction of Arctic species.From about 0.7 Ma onwards, both MDE curves decreased Fig. ; Table 129. Our biogeographic analyses with 13 predefined regions show that Arctic species are derived from multiple sources, in agreement with previous results29. Most biotic dispersals of the Arctic flora were from western North America , whereas there were far fewer dispersal events from other regions  across the angiosperm tree of life . We included 3626 species, of which 548 are distributed in the Arctic and 40 are endemic (restricted to the Arctic). These selected taxa belong to 10 orders and 16 families  between the plastid and nuclear trees52. After removing sixty-eight species, all of which are non-Arctic . We used fossils or secondary ages as calibration points, depending on the information available for each clade (see details in Supplementary Note).For each clade, we performed sequence alignments for each locus in MAFFT v.7.03755 under the dispersal-extinction-cladogenesis model56. To minimize the uncertainties associated with phylogenetic inference, we randomly sampled 1000 posterior trees subsampled from the BEAST analysis as a “trees file” and used the maximum clade credibility (MCC) tree as a final representative tree. To infer the differences between the dynamics of dispersal-associated diversification versus in situ speciation of the Arctic species, we classified species into either Arctic , non-Arctic (B), or occurring in both regions (AB), based on their current known distribution. To examine the possible sources of the Arctic species, we also coded 13 biogeographic regions based on the floristic division of Takhtajan57: (A) Arctic, (B) North Europe, (C) Mediterranean region, (D) North Asia, (E) Qinghai-Tibet Plateau, (F) East Asia, (G) western North America, (H) eastern North America, (I) Africa, (J) Turkey-Iran Plateau; (K) Southeast Asia, (L) South America, and (M) Oceania , closed habitat , or occurring in both habitats. To take into account phylogenetic uncertainty, the MCC tree and 1000 posterior trees subsampled from BEAST analysis were imported as input files for each clade. All analyses were run for 5×106 generations under the fixed JC + G model, with 10 simultaneous chains, sampling every 1000 generations.Ancestral habitats were reconstructed for each clade with endemic Arctic species using the Bayesian binary MCMC (BBM) method in RASP 4.259, “dispersal event into the Arctic” and “in situ diversification event”. To infer general patterns of these two events, we estimated the maximal number of observed dispersal events per Ma (MDisE)60 and in situ diversification events per Ma (MDivE)59, respectively. These MDE rates were calculated by summing potential dispersal or in situ diversification events over all data sets through time using time slices of 0.1 million years , we categorized the species-level biogeographic events for the arctic species on the phylogenetic tree  to determine the optimal model. The 95% confidence intervals of the change points were calculated using the R package lines.segmented function.To assess the 95% confidence intervals of the MDE origination and peaks, we re-sampled 1000 bootstrap pseudoreplicates from the raw credibility intervals (used for inferring the MDisE and MDivE rates above) using the R package Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary


Culturing respiratory epithelial cells at an air–liquid interface (ALI) represents an established method for studies on infection or toxicology by the generation of an in vivo-like respiratory tract epithelial cellular layer. Although primary respiratory cells from a variety of animals have been cultured, an in-depth characterization of canine tracheal ALI cultures is lacking despite the fact that canines are a highly relevant animal species susceptible to various respiratory agents, including zoonotic pathogens such as severe acute respiratory coronavirus 2 (SARS-CoV-2). In this study, canine primary tracheal epithelial cells were cultured under ALI conditions for four weeks, and their development was characterized during the entire culture period. Light and electron microscopy were performed to evaluate cell morphology in correlation with the immunohistological expression profile. The formation of tight junctions was confirmed using transepithelial electrical resistance (TEER) measurements and immunofluorescence staining for the junctional protein ZO-1. After 21 days of culture at the ALI, a columnar epithelium containing basal, ciliated and goblet cells was seen, resembling native canine tracheal samples. However, cilia formation, goblet cell distribution and epithelial thickness differed significantly from the native tissue. Despite this limitation, tracheal ALI cultures could be used to investigate the pathomorphological interactions of canine respiratory diseases and zoonotic agents. The respiratory tract is in close and continuous contact with the outer environment. Through the gaseous exchange, myriads of airborne pathogens directly get in touch with the respiratory epithelium . To prevBordetella bronchiseptica -S-phenylglycine t-butyl Ester (DAPT) can enhance the percentage of ciliated cells in mouse and human airway epithelia [The presence of ciliated cells is essential, seeing as ciliary beating together with the mucus layer produced by secretory cells serves as a primary defense mechanism against pathogens through the mucociliary clearance in vivo ,41. A mu amounts . Compare amounts . Another amounts . Accordi amounts ,50,51,52pithelia . Therefopithelia .The presence of goblet cells in the ALI cultures of this study was confirmed using alcian blue staining, with few positive cells until day 21 under ALI conditions and increasing amounts thereafter. However, alcian blue-stained goblet cells of the present study differed in shape and localization within the epithelium in comparison to their in vivo counterparts. This discrepancy between in vitro and in vivo goblet cells has not been described so far and is most likely due to different culture conditions. Goblet cells can be defined as specialized epithelial cells responsible for the production of mucus, which is then secreted into the lumen of large and small airways ,55. ThisThe transepithelial electrical resistance (TEER) measurement is a quantitative method to evaluate the integrity of endothelial and epithelial monolayers characterized by the development of tight junctions . In caniThe total cell count of ALI cultures in the present study reached comparable levels to the cell count in native tracheas at day 21, implicating that the proliferation of cultured cells is likely sufficient for further use at this time point. The thickness of the entire cultured epithelium increased throughout the culture period and surpassed the native tracheal tissue at day 14. This expansion of thickness might be due to morphological aberrations between cultured and native epithelial cells: the latter might include an increased number of intracytoplasmic vacuoles, increasing the overall cell size and, therefore, the layer thickness of the epithelium under ALI conditions. Additionally, a mild disarrangement of cultured cells was seen throughout the whole epithelium, leading to variations in the number of cell layers and, therefore, in epithelial thickness. This result was surprising as in other previous studies, the thickness of the cultured epithelium was reduced compared to the epithelium in native tissue ,48,63. TOverall, primary tracheal epithelial cultures derived from dogs were successfully established and cultured for up to 28 days after initiation of ALI conditions, resulting in a well-differentiated, pseudostratified epithelium. Using histological, immunohistochemical and electron microscopic methods, we determined that most characteristic features of the respiratory tracheal epithelium in vivo were maintained in vitro, even though the cellular composition and morphology varied between the native tissue and cell cultures. The latter include lack of cilia formation, the orientation of goblet cells in the late phase of ALI cultures and epithelial thickness. These limitations should be taken into account when planning potential in vitro experiments, considering the objective to be investigated. Despite the fact that the developed canine ALI cultures represent a very useful tool for many investigations, further improvements of the described in vitro model may allow applying primary respiratory cells cultured at an ALI as an even more valuable tool for future investigations to study pathogen cell tropism, spread and replication also in regard to the 3Rs principle.Samples from a total of twelve dogs were collected for this study in order to obtain formalin-fixed, paraffin-embedded tracheal tissue as well as primary respiratory epithelial cells for further culture. Five canine tracheas were obtained from necropsy cases at the Department for Pathology, University of Veterinary Medicine, Foundation, Hannover, from two male and three female dogs of different breeds, between two months and 13 years of age, free of respiratory diseases. Additional tracheal tissue from four male and three female, 9 months old Beagle dogs was used. Tissue from the Beagle dogs originated from animals that served as healthy control dogs in regulatory animal studies. The regulatory studies were performed in an Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC)-approved laboratory in accordance with the German Animal Welfare Act and the effective European Council Directive. The studies were approved by the local authorizing agency for animal experiments  on 18 October 2017, as referenced by the approval number 23 177-07/G 17-3-080. For further details regarding available samples from the individual animals, see g for 10 min at 4 °C. The supernatant was discharged, and the cell pellet was resuspended in 50 mL PBS-, followed by centrifugation. The cell pellet was then resuspended in BEGM and filtered through a 100 µm porous mesh. Afterward, tracheal epithelial cells were immediately seeded on, depending on pellet size, one or two collagen-I-coated T75 culture flasks and incubated at 37 °C in a humidified 5% CO2 incubator. The culture medium was supplemented with additional antibiotics   with 250 µL ALI medium in the apical and 500 µL ALI medium in the basal compartment. The cultures were incubated at 37 °C in a humidified 5% CO2 incubator. After 24 h, the medium was removed and cells were washed twice with Hanks’ Balanced Salt Solution (HBSS). Regular medium changes were performed every other day with 200 µL of ALI medium in the apical and 500 µL in the basal compartment. The protocol for the isolation of primary tracheal epithelial cells was adapted and slightly modified as previously described . Freshlyibiotics  and chanI medium  and coun2 incubator, and the ALI medium was changed every second day, with weekly washing steps with HBSS.After seeding, canine tracheal epithelial cells were cultured in submerged conditions on inserts, with daily control measurements of the transepithelial resistance (TEER) as previously described . When TE® ERS-2  was used to verify the integrity of cellular barriers. The chopstick electrode pair was calibrated in ALI medium and placed upright with one electrode in the apical and one in the basal compartment, separated by the layer of cells on the semipermeable membrane. For the measurement procedure, the compartments of two inserts of each dog from a total of six dogs were filled with 500 µL ALI medium in the lower and 200 µL ALI medium in the upper compartment. The resistance across the semipermeable membrane and cell layers  was measured in units of Ω daily after seeding until ALI conditions were established. After that, TEER values were measured once a week for up to 28 days post-initiation of ALI conditions. Semipermeable membranes without a cellular monolayer were measured for blank resistance (Rblank). The tracheal epithelial cell-specific resistance (Rtissue) was calculated as follows: Rtissue (Ω) = Rtotal − Rblank. TEER values are typically given in units of Ω per cm2. Therefore, Rtissue was multiplied by the specific area of the semipermeable membranes used in this study, (0.3 cm2).For TEER measurements, previous protocols  were adaFor histological examination by light microscopy, native tracheal tissue and inserts with tracheal epithelial cells, which were cultured under ALI conditions, were immersion-fixed at day 0, 7, 14, 21 and 28 in culture in 4% paraformaldehyde (PFA) for at least 24 h and routinely embedded in paraffin wax. For further processing, 2–4 µm thick serial sections were cut from the paraffin wax blocks and routinely stained with hematoxylin and eosin (H&E). To visualize mucopolysaccharides in goblet cells, we stained sections with alcian blue as described previously . BrieflyAdditionally, intracellular mucus was stained with periodic acid-Schiff (PAS) reaction after Mc Manus using standard staining protocols . The depFor staining procedures with mucicarmine, the sections were immersed in hematoxylin  for 5 min. For the mucicarmine stain, a stock solution was prepared with 1 g carmine , 0.5 g aluminum chloride , 2 mL distilled water and 100 mL 50% ethanol. The stock solution was diluted 1:10 with 70% ethanol as a working solution in which the sections were stained for 10 min.Immunohistochemistry of PFA-fixed, paraffin-embedded tracheal tissue and tracheal epithelial ALI cultures were performed to characterize the epithelial architecture further using the avidin–biotin–peroxidase complex  method as previously described . The cytImmunofluorescence staining was performed with anti-zonula occludens-1 (ZO-1)  to detect tight junctions. Therefore, the inserts were fixed in 4% PFA at room temperature for 20 min. After blocking with 10% goat serum, inserts were placed in 25 µL antibody solution, and 50 µL antibody solution was pipetted in the apical compartment. After an incubation time of 2 h at room temperature, inserts were washed using PBS- with 0.25% TritonX-100 and incubated using the secondary antibody Alexa Fluor 488 goat–anti-rabbit in the same way as with the primary antibody for 2 h in the dark. The nuclei were stained using FluoroshieldTM mounting medium  and coverslips. The tissue sections were observed with an Olympus IX70 immunofluorescence microscope, and images were captured with an Orca flash 4.0 camera.At each time point, four sections per dog generated from two ALI cultures were evaluated. For the native tracheal tissue, one cross-section was analyzed completely. Briefly, five randomized fields at a 200× magnification were investigated per section using an Olympus BX51 light microscope and photographed with an Olympus DP72 camera. Based on the captured pictures, the total cell count in the H&E staining, alcian blue stained cells, as well as α-tubulin-positive cells were counted, and the epithelial thickness in the H&E staining was measured using the QuPath-0.3.0 software. For quantification of the number of goblet cells present in each sample, respective slides stained with alcian blue solution were evaluated instead of slides stained with Muc5AC antibody because of the observed improved depiction of individual cells. The percentages of alcian blue-stained goblet cells and the α-tubulin-positive ciliated cells were calculated based on the total cell count in both the ex vivo tracheal tissue and the in vitro primary epithelial cell cultures.As previously described  and accoCultures were processed for transmission electron microscopy as previously described . ALI culp-value p < 0.05.For statistical analysis, GraphPad Prism software version 9.0.0 was used. Data obtained from the histological evaluation were analyzed for normal distribution with the Shapiro–Wilk test. Significance was analyzed by one-way-ANOVA and Tukey multiple comparison test. Data were given with standard deviation, and differences are indicated via