Guidelines recommend that all patients with upper gastrointestinal bleeding (UGIB) undergo endoscopy within 24 h. It is unclear whether a subgroup may benefit from an urgent intervention. We aimed to evaluate the influence of endoscopic hemostasis and urgent endoscopy on mortality in UGIB patients with high-risk stigmata (HRS).Consecutive patients with suspected UGIB were enrolled in three Japanese hospitals with a policy to perform endoscopy within 24 h. The primary outcome was 30-day mortality. Endoscopic hemostasis and endoscopy timing  were evaluated in a regression model adjusting for age, systolic pressure, heart rate, hemoglobin, creatinine, and variceal bleeding in multivariate analysis. A propensity score of 1:1 matched sensitivity analysis was also performed.n = 769) was 3.0 h  and early endoscopy (n = 117) was 12.0 h . Successful endoscopic hemostasis and urgent endoscopy were significantly associated with reduced mortality in multivariable analysis . In a propensity-score-matched analysis of 115 pairs, adjusted comparisons showed significantly lower mortality of urgent vs early endoscopy .HRS were present in 886 of 1966 patients, and 35 of 886 (3.95%) patients perished. Median urgent-endoscopy time (A subgroup of UGIB patients, namely those harboring HRS, may benefit from endoscopic hemostasis and urgent endoscopy rather than early endoscopy in reducing mortality. Implementing triage scores that predict the presence of such lesions is important. Gastrointestinal bleeding is a major cause of hospital admissions, incurring significant economic burden to healthcare systems, accounting for >500,000 hospital admissions, 2.2 million days of hospitalization, and $5 billion in direct costs according to a 2018 report in the USA . The abiAlthough endoscopic hemostasis of HRS significantly reduced recurrent bleeding and the need for urgent intervention and surgery, it is unclear whether performing endoscopic hemostasis leads to reduced mortality . FurtherIn this study of patients suspected of having UGIB, we aimed to evaluate the influence of endoscopic hemostasis on overall mortality and identify the optimal timing of endoscopy in patients with HRS.All consecutive adult patients with a suspected UGIB were enrolled at three acute care hospitals in Japan . Although all clinically suspected cases of UGIB were included , we excluded patients with post-procedural UGIB . The institutional review board of each hospital approved the study (20130069). Informed consent or an opt-out clause was obtained from each patient.The primary outcome was the all-cause mortality within 30 days after a suspected UGIB. We divided patients into two groups: those found to have HRS on endoscopy and those without HRS. One of the following conditions was defined as HRS: (i) when the source of bleeding was peptic ulcer disease, spurting, gushing, oozing bleeding, or non-bleeding visible vessel  was seen ; (ii) whThe determination of HRS was adjudicated by showing the endoscopic images of all encountered lesions to experienced endoscopists who were blind to the clinical information of patients. The cause of death, when it occurred, was captured in the medical record.To achieve endoscopic hemostasis, one of or a combination of the following techniques was implemented: argon plasma coagulation, electrocautery, clips, band ligation, epinephrine injections, or sclerosants injection. An urgent endoscopy was defined as one performed within 6 h after the presentation of suspected UGIB, while early endoscopy was done >6 hours of presentation , 17. We t-test or the Mann–Whitney U test. The best cut-off value of endoscopy timing for mortality was calculated by a receiver-operating characteristic (ROC) curve with the greatest sensitivity −(1− specificity). The odds ratio (OR) and 95% confidence interval (CI) of endoscopic hemostasis and endoscopic timing were calculated using univariable and multivariable logistic-regression analysis.Categorical variables are expressed in number and percentage, and were compared using the chi-square test or Fisher's exact test. Quantitative numerical variables are presented by mean and standard deviation (SD) or median and interquartile range (IQR), and were compared using Student’s n = 50). The continuous variables in confounding factors were classified into binary categorical variables according to the existing cut-off in the literature or ROC. It means that we used the cut-off of AIMS65 for age (≥65 years), the cut-offs of GBS for systolic blood pressure (SBP) (<90 mmHg) and heart rate (HR) (≥100 beats/min), and the cut-off of the randomized–controlled trial (RCT) in the New England Journal of Medicine for hemoglobin level (<7.0 g/dL), but the cut-offs for creatinine (≥1.0 mg/dL) were not available in the past literature, so we calculated the optimal cut-off from our current data [Three multivariable logistic-regression models with different confounding factors were used to demonstrate robustness because the number to be adjusted for as a confounding factor was limited due to the small number of mortality events  and early (>6 h) endoscopy groups in patients with HRS . Propensp-value <0.05 was considered statistically significant. All analyses were done using JMP(r) statistical software version 14.1.0 .As sensitivity analyses, time cut-offs (≤6 vs 6–12 vs >12 h) , 23, 24,The study flow is shown in Among 1,966 patients, 886 had HRS, of whom 35 (3.95%) patients perished (19 events were related to bleeding and 16 to other causes). In this group of patients with HRS, the relationship between endoscopic timing and mortality was assessed, and the best cut-off value to achieve the lowest mortality was 5.5 h. The median time of urgent endoscopy (≤6 h) group was 3.0 h  in 769 patients and that of the early endoscopy (>6 h) group was 12.0 h  in 117 patients. Endoscopic hemostasis and urgent endoscopy were significantly associated with lower mortality in univariable analysis . After aP < 0.001).After propensity-score matching, 115 matched pairs were generated from 769 patients in the urgent-endoscopy group and 117 patients in the early-endoscopy group in a 1:1 manner. There were no significant differences in characteristics between the two matched groups . The morP = 0.021), while endoscopy 6–12 h was not . In multivariable logistic analysis (model 1), endoscopy ≤6 h was significantly associated with lower mortality  compared with endoscopy >12 h, while there was no significant difference in mortality between two groups (6–12 and >12 h groups)  . Endoscopy ≤6 h was significantly associated with lower mortality than endoscopy >12 h in univariable analysis  .P = 0.34, and OR, 2.62; 95% CI, 0.31–22.1; P = 0.35, respectively). In the multivariable logistic analysis, endoscopy ≤6 and 6–12 h compared with >12 h were not significantly associated with mortality   was not related to bleeding, but was due to other causes . Neither endoscopic hemostasis nor urgent endoscopy was significantly associated with reduced mortality in both univariable and three models of multivariable logistic analysis . Among tctively) .P = 0.44), while in the non-variceal-bleeding-with-HRS group, urgent endoscopy was significantly associated with lower overall mortality .Of 886 patients with HRS, 143 patients had variceal bleeding and 743 had non-variceal bleeding . In the P = 0.18), it was significantly associated with decreased UGIB-specific mortality in multivariable logistic-regression analysis  . After adjusting for confounders (model 1) in multivariable logistic analysis , althoug = 0.04) .Of 730 patients with GBS ≥12, HRS was found in 427 (58.5%). After adjusting for confounders (model 1) in multivariable logistic analysis, urgent endoscopy was not significantly associated with mortality and UGIB-specific mortality.Of 389 patients with SBP <90 mmHg (hemodynamic instability), HRS was found in 279 (71.7%). After adjusting for confounders (model 1) in multivariable logistic analysis, urgent endoscopy was not significantly associated with mortality and UGIB-specific mortality.P-value of the propensity-matching score is 0.047, the results are likely robust and unlikely attributed to chance because all analyses showed a significant difference in the influence of urgent endoscopy significantly reducing mortality. In a subgroup analysis, urgent endoscopy was significantly associated with lower mortality rates in patients with non-variceal bleeding. Although urgent endoscopy has been recommended for patients with variceal bleeding with HRS, this result may support that the patients with non-variceal bleeding may also benefit from urgent endoscopy when HRS are suspected to be present [In this large multicenter cohort of Japanese patients with suspected UGIB, we found that performing endoscopic hemostasis and urgent endoscopy (≤6 h from presentation) were factors associated with lower overall mortality compared with early endoscopy (>6 h), but only in those patients later found to harbor HRS. In contrast, those patients without HRS did not benefit from this urgent intervention. In addition, the mortality rate was significantly lower with urgent endoscopy either in multivariate logistic-regression analysis or in the propensity-score-matched analysis. Although the  present .HRS are defined by well-established endoscopic findings indicating the need for endoscopic hemostasis by international consensus statements . To the To date, there are no RCTs designed to randomly allocate endoscopy timing in UGIB patients based on their likelihood of harboring HRS . NonetheThe HARBINGER, which has been recently validated, is the first simple score incepted to predict the presence of HRS , 13. TheOur study has several shortcomings, inherent in the lack of randomization and the restriction of our demographics to Japanese patients. To ascertain whether mortality is truly reduced as a result of urgent intervention in patients with HRS, a powered RCT is needed in which patients are randomized according to the HARBINGER. However, ethical considerations may not permit the randomization of such high-risk groups.In addition, it is almost certain that there are other confounders not controlled for our analyses. For example, we were unable to calculate for the American Society of Anesthesiologists score for each patient as underlying conditions and the size/number of ulcers was not described in our database. We attempted to compensate for such shortcomings by adjusting and controlling for not only the confounding factors identified from the analysis of this study data, but also those identified in the literature, including congestive heart failure and any hepatic disease as important underlying diseases in UGIB and by other offered treatments . Further, we were able to describe the etiology of UGIB  accurateIn conclusion, both endoscopic hemostasis and urgent endoscopy within 6 h from the presentation were significantly associated with lower all-cause mortality in patients who harbored HRS compared with early endoscopy (conducted >6 h) in a large multicenter Japanese cohort. In patients with HARBINGER ≥2, urgent endoscopy was significantly associated with reduced mortality from UGIB. Future studies should investigate whether UGIB patients’ triage, according to the HARBINGER, will accrue a mortality benefit when urgent endoscopic hemostasis is pursued.M.H., E.I., Te.K., J.M, T.M, N.H., Y.O., S.N., Y.H., H.O., and Ta.K. conceived of and designed the project; M.H., E.I., F.B., Te.K., and J.M analysed and interpreted the data; M.H., E.I., F.B., and J.M drafted the manuscript; M.H., E.I., F.B., Te.K., J.M, K.M, S.F., T.M, N.H., and Ta.K. made critical revisions to important intellectual content. All authors read and approved the final manuscript.None.


Diabetes mellitus type two is one of the major cardiovascular risk factors. Treatment of diabetes can reduce this risk, but the treatment options differ a lot in their risk-reducing capabilities. We compared the impact of insulin degludec (IDeg-100) and insulin glargine U300 (IGlar-300) on cardiovascular risk parameters - glycaemic variability (GV), arterial stiffness and lipid parameters - in insulin naive patients with DMT2.To 23 individuals who previously had uncontrolled DMT2 on two or more oral antidiabetic drugs, IGlar-300 and IDeg-100 were applied for 12 weeks and then switched in a cross over design manner. Prior and after of each insulin phase, we analysed biochemical parameters,7-point SMBG profile over three days and arterial stiffness which was assessed indirectly by measuring the augmentation index (AIx) on the principles of applanation tonometry.p = 0.0215).There were no significant differences between IGlar-300 and IDeg-100 regarding reduction of mean glucose values and coefficient of variation (CV). Both insulins insignificantly reduced AIx for standardised pulse of 75 beats/min and without differences between them. IGlar-300 and IDeg-100 reduced triglycerides and increased HDL with no significant difference between the two insulins. IGlar-300 increased the total cholesterol level and IDeg-100 decreased total cholesterol, but without statistically significant difference. IGlar-300 increased LDL level by 0.508 mmol/L and IDeg-100 decreased LDL by 0.217 mmol/L, with statistically significant difference (This study did not show significant difference between IGlar-300 and IDeg-100 regarding glycaemic parameters and augmentation index using the same dose of 0.2 IU/kg for both insulins, but it has revealed possible differences in impact on lipid profile.NCT04692415. Retrospectively registered on December 31th 2020.Clinicaltrials.gov, The online version contains supplementary material available at 10.1186/s12902-021-00746-1. Although the pathogenesis of atherosclerosis is mediated through multiple and complex mechanisms, including lipoprotein abnormalities  × 100 . The AIxThe number of subjects to include in the protocol was selected according to the previous available literature , 28. StaAll patients  completed the trial, 23 were analysed. No unexpected events, harms or unintended effects of therapy were observed. Their mean basal values are given in Table p = 0.06), and without significant difference between the two insulins (p = 0.17)  and in comparison of the two insulins there was no statistically significant difference (p = 0.07)  of glucose excursions was 0.36 for IGlar-300 and 0.06 for IDeg-100 which was insignificant p = 0. and in cp = 0.22). When compared, CV for these two insulins was not significantly different (p = 0.20)  on the first day was 0.37 (37 %) for IGlar-300 and 0.21 for IDeg-100 which was statistically insignificant (p = 0.08), and without significant difference between the two insulins (p = 0.07)  and when we compared the SD of the two insulins there was no significant difference (p = 0.17) . When compared, CV for the two insulins was no significantly different (p = 0.08)  for IGlar-300 and 0.04 for IDeg-100 and that was statistically insignificant (p = 0.08), again without significant difference between the two insulins (p = 0.20)  and comparison of SD of the two insulins revealed no statistical difference (p = 0.14)  day of the SMBG, the insulins reduced mean glucose levels by 0.04 (IGlar-300) and by 0.11 mmol/L (IDeg-100) which was statistically insignificant  . There was no significant difference between the two insulins  . When compared, there was no difference in AIx between the two insulins (p = 0.06)  Fig. ..p = 0.4275). IGlar-300 increased the total cholesterol level by 0.317 mmol/L and IDeg-100 decreased total cholesterol by 0.295 mmol/L, but without statistically significant difference (p = 0.0813). Both insulins increased HDL level – IGlar-300 by 0.1 mmol/L, and IDeg-100 by 0.065. There was no significant diffference in impact on HDL (P = 0.4015). IGlar-300 increased LDL level by 0.508 mmol/L and IDeg-100 decreased LDL by 0.217 mmol/L, with statistically significant difference (p = 0.0215)  particles and chylomicrons from the serum with consequent transportation of fatty acids to the fat tissue. Insulin also affects adipocytes by boosting triglyceride synthesis and the skeletal muscles by lowering the activity of lipoprotein lipase. The latter can intercept the accumulation of lipids in the muscles. Finally, insulin hinders hormone sensitive lipase in fat tissue, decreasing lipolysis in that way. The total result of insulin impact on the metabolism of the lipids is a decrease of circulating triglycerides and triglyceride-rich lipoprotein concentration .There are very few studies depicting the impact of specified insulin formulations on lipid profiles.As the induction of insulin in the treatment of diabetic patients has shown also a beneficial effect on glucose variability and arterial stiffness , 10, theFurthermore, as insulin degludec and glargine U300 came into the market practically at the same time, the comparison of these two last-generation basal insulin analogues became unavoidable. As it is stressed in the Introduction, there are a number of studies comparing degludec and glargine U300, but not in the manner designed in this study.The results of our study showed significant difference between these two insulins in LDL concentrations with unexpected failure of IGlar300 to reduce it. Although without statistical significance, there were signals that there could be more differences in other lipid parameters. These results should be confirmed in the studies with much larger number of subjects, longer duration and higher doses of insulins. Comparison with other basal insulins, and even short acting insulins, should be performed. Possible mechanisms through which potential differences occur, also need to be explained. Perhaps the different PK/PD profiles of different insulins can play a role in the degree of LDL receptor activation  or in plThe present study did not show significant differences in impact of IDeg100 and Iglar300 on glycaemic variability and arterial stiffness using the same dose of 0.2 IU/kg for both insulins. If we neglect the possibility that these two insulins simply are not significantly different, still there are several possible reasons for the lack of the difference.Firstly, we had a relatively small number of subjects. Secondly, three months of exposure to each insulin could be regarded as relatively short period , 41, altWe applied both insulins in the standard initiating dose of 0.2 IU per kg of body weight. As possible hypoglycaemia could have a strong impact on the results of glycaemic variability and oxidative stress, we decided not to up-titrate the dose to the end of observed period. Several studies have shown that hypoglycaemia can decrease nitric oxide and cause the “reperfusion like” effect, influencing the level of oxidative stress in that way –21. AlthTo conclude, in this setting, IDeg-U100 and IGlar-U300 showed no significant difference in impact on the glycaemic variability and arterial stiffness using the same dose of 0.2 IU/kg for both insulins. We noticed significant difference on LDL levels with signals for potential differences in other lipid parameters. Thus, further investigations are needed to either confirm or oppose these findings.Additional file 1.


The incidence rates of thyroid tumors and nodular goiter show an upward trend worldwide. There are limited reports on the risk of perchlorate and iodine on thyroid tumors, but evidence from population studies is scarce, and their impact on thyroid function is still uncertain. Therefore, the objective of this study was to investigate the association of perchlorate and iodine with the risk of nodular goiter (NG), papillary thyroid microcarcinoma (PTMC), and papillary thyroid carcinoma (PTC) and to assess the correlation between perchlorate and iodine with thyroid function indicators.A case–control population consisting of 184 pairs of thyroid tumors and nodular goiter matched by gender and age (±2 years) was recruited in this study. Serum and urine samples were collected from each participant. Thyroid function indicators in serum were tested by automatic chemical immunofluorescence, and perchlorate and iodine levels in urine were determined by ultra-high performance liquid chromatography tandem-mass spectrometry and inductively coupled plasma-mass spectrometry, respectively. Conditional logistic regressions and multiple linear regressions were used to analyze the associations.P < 0.05). Perchlorate was positively associated with PTC  in a non-linear dose–response relationship, but there was no association between perchlorate and NG or PTMC. Iodine was not associated with the risk of thyroid tumors and NG and did not correlate with the thyroid function indicators. Furthermore, perchlorate showed a positive correlation with thyroid stimulating hormone (TSH) at iodine adequate levels (P < 0.05), and a negative correlation with free triiodothyronine (FT3) and a positive correlation with thyroglobulin antibody (TgAb) at iodine more than adequate or excess levels (P < 0.05).Urinary perchlorate concentration was significantly higher in total cases, NG and PTC than in the corresponding controls (Perchlorate can increase the risk of PTC in a non-linear dose–response relationship and disturb the thyroid hormone homeostasis and thyroid autoantibody levels.The online version contains supplementary material available at 10.1186/s12940-021-00818-8. Patients with thyroid tumor have higher perchlorate exposure than control.Perchlorate can increase the risk of papillary thyroid carcinoma.Perchlorate can disturb the homeostasis of thyroid function.Perchlorate was positively correlated with thyroglobulin antibody.The online version contains supplementary material available at 10.1186/s12940-021-00818-8. The incidence of thyroid tumor has been sharply growing around the world, identification and characterization the risk factors for thyroid cancer and nodules have become a scientific hot topic in recent years. Thyroid tumors are divided into benign thyroid adenoma and malignant thyroid cancer (TC), and thyroid nodules (TNs) are a common benign proliferative disease of the thyroid gland with the detection rate in the general population as high as 65% . Studies− on plasma membrane glycoprotein, thereby preventing I− from entering thyroid follicular cells and interfering with the key steps of thyroid hormone biosynthesis [It has been suggested that multiple factors, including genetics, radiation, iodine, autoimmune diseases, and complex and diverse environmental factors, such as environmental chemicals, may be the risk factors for thyroid tumors . It has ynthesis . Howeverynthesis –20. Furtynthesis . HoweverTherefore, exploring the effects of perchlorate and iodine on three different pathological types of thyroid tumors and on thyroid function is urgently needed. The purpose of the present study was to investigate the effect of perchlorate and iodine on the risk of nodular goiter (NG), papillary thyroid microcarcinoma (PTMC), and PTC and to investigate the correlation with thyroid function indicators, so as to identify the key risk factors for thyroid disease. The study will provide a scientific basis for the prevention and control of thyroid tumors in future.This study recruited 184 patients with thyroid tumors and NG and 211 controls among total 395 participants, of which 184 case–control pairs were matched by gender and age (± 2 years). The patients with newly diagnosed thyroid tumors and nodular goiter, not undergoing radiotherapy and chemotherapy, from September 2018 to September 2019 were recruited at a specialist clinic from two comprehensive third class hospitals in Shenzhen. They included 72 patients with NG, 46 patients with PTMC and 66 patients with PTC. All the three pathological types categorized as total cases. These patients met the “NCCN Clinical Practice Guidelines in Oncology: Thyroid Carcinoma” diagnostic guidelines issued by the National Comprehensive Cancer Network (NCCN) . All parFasting venous blood was collected from the participants, and serum was separated by centrifugation at 3000 rpm for 15 min within 2 h and stored in cryogenic vials. The morning fasting middle-segment urine was collected and transferred into centrifuge tubes for storage. All samples were stored at − 80 °C in an ultra-low temperature refrigerator until analysis.Serum levels of thyroid function indicators, including thyroid stimulating hormone (TSH), triiodothyronine (TT3), thyroxine (TT4), free triiodothyronine (FT3), free thyroxine (FT4), thyroid peroxidase antibody (TPOAb), and thyroglobulin antibody (TgAb), were measured by an automatic chemiluminescence analyzer . The reference range appears below: TSH: 0.30-4.50 μIU/mL; TT3: 0.69-2.15 ng/mL; TT4: 52.00-127.00 ng/mL; FT3: 2.00-4.20 pg/mL; FT4: 8.90-17.20 ρg/mL; TgAb: 0.00-95.00 IU/mL; TPOAb: 0.00-30 IU/mL .Perchlorate in urine was detected by high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). Specifically, 570 μL of urine sample was added into a 2-mL centrifuge tube; 30 μL of 1 μg/mL perchlorate isotope internal standard was added, mixed, and passed through 0.2-μm filter membrane. Finally, 5 μL of the solution was injected into the Infinity Lab Poroshell 120 HILIC-Z column . The standard substance of perchlorate was purchased from Beijing North Weiye Institute of Metrology and Technology , and the internal standard substance was purchased from Shanghai ZZBIO Co. Ltd. . Two quality control samples were detected for every 20 samples, and the spiked recovery rate for samples was verified according to the low, medium, and high concentration of the samples. The limits of detection and quantification were 0.083 and 0.275 μg/L, respectively, and the spiked standard recovery was found between 93.2 and 115.5%.The level of iodine in urine was evaluated by inductively coupled plasma mass spectrometry . First, 1 mL of urine sample was accurately added into a 15-mL centrifuge tube, diluted to 10.0 mL with 0.25% tetramethylammonium hydroxide solution, oscillated on the vortex mixer, and then injected onto the ICP-MS. The standard substance for the analysis of iodine composition in freeze-dried human urine  was used as the quality control sample to control the whole process of detection.c: corrected concentration; Cd: actual detected concentration; USGm: the median of USG in urine of case or control group; USGd: actual detected USG in individual urine):The levels of perchlorate and iodine in urine were corrected by urinary specific gravity (USG), and the formula is as follows   and R 3.6.3  were used for statistical analysis of the data. Categorical variables were compared by the chi-square test. Mean ± SD (standard deviation) or median (interquartile range) was used to describe continuous variables. The Wilcoxon signed-rank test was used for intergroup comparisons, and the Kruskal–Wallis test was used for comparison of multiple groups.P ≤ 0.10 as the inclusion criterion were conducted, and body mass index (BMI), smoking, and drinking were included as confounding variables. Restricted cubic spline regression with three knots  was used to evaluate the dose–response relationship between perchlorate exposure and the risk of thyroid tumors and NG. The relationship between perchlorate, iodine, and thyroid function indicators was analyzed only in the controls. Spearman’s rank correlation and multiple linear regression analysis were used to evaluate the relationship between urine target variables (perchlorate and iodine) and TSH, TT3, TT4, FT3, FT4, TPOAb, and TgAb in the control group. The variables with non-normal distribution  were natural log-transformed. Multiple linear regression model adjusted for gender, age, BMI, smoking, and drinking was established. All statistical significance level was set at P < 0.05 (two-sided).Conditional logistic regression was used to assess the risk of thyroid tumors and NG related to urinary target variables  by using crude and adjusted odds ratios (ORs) and their corresponding 95% confidence intervals (CIs). According to WHO standards, iodine nutrition levels were divided into insufficient (< 100 μg/L), adequate (100–200 μg/L), more than adequate (200–300 μg/L), and excess (≥300 μg/L). Multiple conditional logistic regression analyses with P < 0.05).Population characteristics are presented in Table P < 0.05). After pathological classification, perchlorate concentrations were higher in NG and PTC than in the corresponding controls (P < 0.05). There was no significant difference among the three pathological types (P > 0.05). Furthermore, the levels of iodine in urine were not significantly different between total cases, NG, PTMC, PTC, and their corresponding controls (P > 0.05).The concentration distribution of perchlorate and iodine in urine is shown in Table P > 0.05). Moreover, there was no significant association between iodine and thyroid tumors and NG (P > 0.05).Furthermore, potential confounding variables for thyroid tumors and nodular goiter have been evaluated   but also belonged to a nonmonotonic trend  Fig. . The risend Fig.  A and B.end Fig.  C.Fig. 1P > 0.05)  and 0.15 , respectively Table . No sign5) Table . Next, tThe present study demonstrated a positive association between perchlorate and the risk of PTC and perchlorate can disturb the homeostasis of thyroid function, but did not find an association between perchlorate and NG and PTMC. In addition, no association was found between iodine and the risk of thyroid tumors and nodular goiter, and thyroid function. The association of endocrine-disrupting compounds and iodide inhibitors with thyroid diseases has received worldwide attention in recent years. However, few population-based studies have examined the relationship between iodination inhibitors and thyroid diseases. To our knowledge, the present study is the first population-based study to simultaneously examine the association of perchlorate exposure with three thyroid neoplasma types .P-non-linear < 0.05). This is consistent with another small population study conducted in China, in which the association of perchlorate exposure and PTC was found  [P75 level in the controls from the present study, which provided a statistical reference for the human exposure threshold. Animals studies have shown that exposure to perchlorate increases the size and number of thyroid follicles, which leads to structural changes in the thyroid gland [Our results showed a positive association between perchlorate exposure and the risk of PTC  with non-linear dose–response relationship (03–5.03) . The ris03–5.03)  includedid gland . Howeverid gland . Moreoveid gland . NG is aid gland . Given tFurthermore, BMI was found to be a protective factor for thyroid tumors and NG, while drinking was a risk factor in this study. Similar findings were reported in Mijović T et al., 2009  and 2010The effects of perchlorate on the indicators of thyroid function were assessed in this study, and the results showed that perchlorate was negatively correlated with FT3 and positively correlated with TgAb, and only positively correlated with TSH when iodine at adequate levels. According to the National Health and Nutrition Examination Survey (NHANES) in the United States, perchlorate was positively associated with TSH only in women aged above 12 years, regardless of whether iodine was adequate or not; in contrast, perchlorate was negatively associated with TT4 only in iodine deficiency status, suggesting an association between perchlorate and hypothyroidism . HoweverBoth TPOAb and TgAb are markers of thyroid autoimmunity and can assist in the prediction of thyroid disease, such as hypothyroidism and prognosis of hyperthyroidism treatment . Our stu− 6 mol/L) and ultra-high dose (1.0 × 10− 3 mol/L) of iodine induced high expression of the SPANXA1 gene and promoted TC cell proliferation and apoptosis [For iodine, we found no association with the risk of thyroid tumors and NG. Wang et al., 2014 pointed out that high iodine levels significantly correlate with the occurrence of benign and malignant thyroid tumors and the aggressiveness of PTC . Howeverpoptosis . Furtherpoptosis . This ispoptosis , 43.Iodine plays an important role in the synthesis of thyroid hormones. However, no correlation between iodine levels and the thyroid function indicators was found in this study. This is similar to a previous cross-sectional study in China, which found no significant association between iodine levels and TSH, FT3, or FT4 . SimilarThe source of perchlorate exposure for the recruited population was analyzed in this study. The median perchlorate concentration in this study (11.27 μg/L) was notably higher than that of 6044 participants of the general population included in the NHANES 3.9 μg/L) ; however μg/L ; hThere are some strengths and limitations in this study. First, cases with three pathological types of thyroid tumor and corresponding controls were matched by gender and age and recruited based on strict inclusion and exclusion criteria. Second, the authenticity and accuracy of data were ensured under strict quality control measures. The confounding variables were adjusted for using a multifactorial model. This study was the first to compare the effects of perchlorate exposure on the three types of thyroid neoplasms. Thus, the findings provide important etiological evidence for thyroid tumor and nodular goiter in order to improve thyroid disease prevention in future. However, there are some limitations to this study. First, the sample size in different pathological types was small, especially in the PTMC group. Second, the levels of perchlorate and iodine in urine were based on a single measurement as it is difficult to reflect long-term human burden levels in a cross-sectional study. However, the present study found the association of perchlorate with PTC and first indicated that perchlorate has no statistical association with the risk of NG and PTMC. In future, population-based cohort studies with precisely monitored iodine intake through long-term follow-up in the exposed populations from perchlorate-contaminated areas should be conducted.We found that perchlorate can increase the risk of PTC in a non-linear dose–response relationship, but no significant associations with NG and PTMC. Moreover, perchlorate can disturb thyroid hormone homeostasis and thyroid autoantibody levels. However, the association between iodine and thyroid tumors, NG, and thyroid function indicators has not been found in this study. The present study provided important population evidence for the identification of the risk factors of thyroid tumors and NG from the exogenous environment chemical exposure.Additional file 1: Table S1. The influencing factors of thyroid tumor and nodular goiter in univariate logistic regression model. Table S2. Dose-response relationship between perchlorate exposure and thyroid tumors and nodular goiter based on restricted cubic spline (RCS). Table S3. Correlation analysis of perchlorate and iodine with thyroid function indicators. Table S4. Distribution of people at different iodine nutrition levels. Table S5. Thyroid autoimmune antibodies concentration in the control group at different iodine nutrient levels.


Enhanced recovery after surgery (ERAS) pathways have been shown to improve clinical outcomes after surgery. Considering the importance of patient experience for patients with benign surgery, this study evaluated whether improved compliance with ERAS protocol modified for gynecological surgery which recommended by the ERAS Society is associated with better clinical outcomes and patient experience, and to determine the influence of compliance with each ERAS element on patients’ outcome after benign hysterectomy.A prospective observational study was performed on the women who underwent hysterectomy between 2019 and 2020. A total of 475 women greater 18 years old were classified into three groups according to their per cent compliance with ERAS protocols: Group I: < 60% (148 cases); Group II:≥60 and < 80% (160 cases); Group III: ≥80% (167 cases). Primary outcome was the 30-day postoperative complications. Second outcomes included QoR-15 questionnaire scores, patient satisfaction on a scale from 1 to 7, and length of stay after operation. After multivariable binary logistic regression analyse, a nomogram model was established to predict the incidence of having a postoperative complication with individual ERAS element compliance.P < 0.001) and length of stay after surgery . Increased compliance was also associated with higher patient satisfaction and QoR-15 scores (P < 0.001),. Among the five dimensions of the QoR-15, physical comfort (P < 0.05), physical independence (P < 0.05), and pain dimension (P < 0.05) were better in the higher compliance groups. Minimally invasive surgery (MIS) (P < 0.001), postoperative nausea and vomiting (PONV) prophylaxis (P < 0.001), early mobilization (P = 0.031), early oral nutrition (P = 0.012), and early removal of urinary drainage (P < 0.001) were significantly associated with less complications. Having a postoperative complication was better predicted by the proposed nomogram model with high AUC value (0.906) and sensitivity (0.948) in the cohort.The study enrolled 585 patients, and 475 completed the follow-up assessment. Patients with compliance over 80% had a significant reduction in postoperative complications (20.4% vs 41.2% vs 38.1%, Improved compliance with the ERAS protocol was associated with improved recovery and better patient experience undergoing hysterectomy. MIS, PONV prophylaxis, early mobilization, early oral intake, and early removal of urinary drainage were of concern in reducing postoperative complications.ChiCTR1800019178. Registered on 30/10/2018.Chinese Clinical Trial Registry, The online version contains supplementary material available at 10.1186/s12871-021-01509-0. Enhanced recovery after surgery (ERAS) pathway is a multimodal perioperative care approach, designed to reduce perioperative stress and shorten recovery time . The sucHysterectomy on benign indication is the most common major gynecological surgery performed worldwide . The aimRecently, enhanced recovery protocols have been introduced in general settings for gynecological surgeries –15. Howe–020), and registered with ClinicalTrials.gov . Patients were informed of the ERAS protocols and signed the informed consent form before study entry, and the study was conducted in accordance with the Declaration of Helsinki. A prospective observational study on patients undergoing benign hysterectomy was performed at the Department of Gynecology in the First Affiliated Hospital of Chongqing Medical University in China during the period February 1st 2019 to December 31st 2020, with the ERAS protocols underwent.This study was approved and conducted by the local Ethics Committee of the First Affiliated Hospital of Chongqing Medical University . Mean total compliance was calculated as the average of all perioperative ERAS interventions. Patients were categorized into three groups by their compliance rates with the ERAS pathway. The same cut-off values as previous studies were used, , 22 wher(PONV), diagnosis and type of surgical procedure were recorded. Preoperative comorbidities were also recorded, such as hypertension, diabetes, chronic obstructive pulmonary disease (COPD), ischemic heart disease and asthma. After entering the operating room, the plasma electrolyte level was monitored by invasive arterial pressure puncture. Preoperative hypokalemia was defined as less than 3.5 mmol/L. Intraoperative data were prospectively collected, including duration of surgery (min), intraoperative net fluid input and blood loss.Characteristics of the study population, age, body mass index (BMI), education level, smoking status, nutritional risk screening (NRS) score, NYHA status, ASA status, history of surgery, preoperative hemoglobin, presence of chronic pain, history of postoperative nausea and vomiting All of the patients were asked to complete 15-item quality of recovery (QoR-15) questionnaire, a widely used self-rated questionnaire for early postoperative quality of recovery , 24 , surgical site infection, and pulmonary infection. Postoperative complications were monitored until 30 days after surgery, which were defined by the guidelines for European perioperative clinical outcome definitions , as showSecondary outcomes included patient satisfaction, QoR-15 scores (including five dimensions), LOS after surgery, mortality, postoperative hospitalization costs, readmission rate within 30 days post-discharge. LOS after surgery was defined as the number of days patients stayed in the hospital after operation. Postoperative hospital costs were presented as RMB converted to Euro (November 2020), which were obtained from hospital databases.The characteristics of study participants in the three groups were described with a descriptive analysis, while the normal distribution of the data was checked with Shapiro-Wilk test. Outliers were investigated and eliminated in the event of a demonstrably incorrect measurement or input error. Categorical variables were described as numbers with percentages, and continuous variables were described as the means with standard deviations (SD) or median and interquartile range (IQR), depending on the distribution, which was checked through visual inspection of the histogram. The Chi square test, repeated measures analysis of variance, Mann-Whitney U test and Kruskal-Wallis test were used to compare the outcomes between groups where appropriate. Fisher’s exact test was used for categorical variables when the number of events was less than five.P value less than 0.05 were included in the multivariable binary logistic regression and hazard ratios of each factors were calculated. Through the multivariable binary logistic regression analysis, the factors with P value less than 0.05 were selected to develop a nomogram prediction model by R software. The discrimination performance of the nomogram model was quantified with receiver operation characteristic (ROC) curve, with the value of the area under the curve (AUC) of the ROC curve between 0.5 and 1.0, and the closer the AUC value is to 1, the better discrimination performance the model has. Calibration curves were plotted to assess the calibration of the nomogram model.Independent of the previous analysis, patients were divided into two groups based on the presence or absence of postoperative complications. All the factors including the clinicopathological factors and each component of ERAS were put into the univariable logistic regression to analyze their correlation with the probability of having any postoperative complication. Then the factors with A sample size calculation was based on the primary endpoint, the incidence of postoperative complications, as the previous study had reported that the incidence of postoperative complications undergoing gynecological operation was approximately 25% [P value < 0.05 was regarded as statistically significant.Statistical analyses were completed in SPSS for Windows version 23.0  and GraphPad Prism for Windows version 8.0 . The nomogram plot, ROC curve and calibration curve were plotted using R software (version 3.6.2) with RMS, ROCR package. A 2-sided A total of 585 women were screened for eligibility from February 2019 to December 2020, of whom, 542 were enrolled . Meanwhile, the intraoperative net fluid input volume of Group III was lower than that of other groups, but the difference was not statistically significant.Furthermore, no clinically significant differences were observed in diagnosis, duration of operation, preoperative hemoglobin or bleeding volume. Significantly, there was a higher proportion of preoperative hypokalemia incidence in the Group I than that in groups II and III . Compared with group I, the incidence of PONV and moderate-to-severe postoperative pain decreased significantly than that in the group III (P < 0.001). The incidence of pulmonary infection was 6.1, 6.3, and 1.8% (P = 0.044). No statistically significant difference in the other complications among the three groups was observed (P > 0.05).Postoperative complications of the three groups were shown in Table P < 0.001). In addition, compare with Group II, the total QoR-15 scores in Group III were higher in the first 3 days after surgery (P < 0.001)  Fig. A.Fig. 2AP < 0.05), physical independence (P < 0.05), and pain dimension (P < 0.05) in the group II and III were significantly higher compared to the group I in the first 3 days after surgery; scores of emotional status (P < 0.05) in the group III was significantly higher than those in the group I and II in the first 2 days after surgery; scores of psychological support (P < 0.05) in the group III were significantly higher compared to the group I and II on POD 2, meanwhile no significant differences in the incidence of no-planned re-operation and postoperative hospitalization cost was observed Table . One loc–4) vs 5(5–6), P < 0.001; I vs III: 4(4–4) vs 6(5–6), P < 0.001; II vs III:5(5–6) vs 6(5–6), P < 0.001). In addition, patient satisfaction was also significantly higher in the groups II and III than that in the Group I on the 30 day after discharge (I vs II: 4(4–5) vs 5(5–6), P < 0.001; I vs III: 4(4–5) vs 6(6–7), P < 0.001; II vs III:5(5–6) vs 6(6–7), P < 0.001).Patient satisfaction on discharge day and the 30 days after discharge in each group was shown in Fig. P values more than 0.05 were excluded from multivariate analysis, including no bowel preparation (P = 0.573), oral carbohydrate loading (P = 0.121), no abdominal drainage (P = 0.062), no routine nasogastric tube (P = 0.339), postoperative glucose control (P = 0.203) and peritoneal drainage (P = 0.215). The other factors with P values less than 0.05, were further included in the multivariable logistic regression. Finally, five factors with P values less than 0.05 in multivariable regression analysis were recruited to construct the prediction model, including minimally invasive surgery (p < 0.001), PONV prophylaxis with over 2 antiemetic agents (p < 0.001), early mobilization (p = 0.031), early oral intake (p = 0.012), and early removal of urinary drainage (p < 0.001).The univariable logistic regression analysis was used to analyze the compliance with each ERAS component that might affect the probability of having any postoperative complication. Table . The facThe nomogram prediction model was established with 5 independent factors to predict the risk of postoperative complications. As shown in Fig. ’s hospitalization, which may adversely influence compliance. Furthermore, implementation of ERAS pathway is associated with a shift in clinical routines, from old practices to new pathway. This study is the first study to our knowledge determining the effect of the compliance of patients to an ERAS protocol after benign hysterectomy on outcomes with the QoR-15 scales and patient satisfaction. Our current study indicated that an overall compliance rate of 80.6% for ERAS in patients undergoing hysterectomy. Similarly, a recent observational study reported a mean compliance rate of 77% after the ERAS program was implemented in gynecologic oncology [Implementation of the ERAS pathway remains an ongoing challenge in clinical practice which requires the engagement of nursing team, surgical team, and patients. Individual ERAS components are implemented at different stages during the patientoncology . Consistoncology , 5, 30, Most previous studies evaluating postoperative recovery have focused on physiological parameters such as LOS and morbidity , 31–34. We found the QoR-15 scores and patient satisfaction were significantly higher in the higher compliance group, which can be explained by the reduction pain and improvement mood during the early postoperative period. In the ERAS protocols, preoperative information education, no prolonged fasting, no bowel preparation and oral carbohydrate loading can relieve the patient discomfort such as anxiety, hungry and thirsty. In addition to the above advantages, it also increases patient comfort and physical independence by reducing the incidence of postoperative nausea and vomiting, relieving pain and accelerating the recovery. The increase in patient satisfaction after multimodal analgesic approaches may be a significant benefit, along with a reduction in postoperative complications. In order to avoid the interference of other factors on the results, we really scored the patients, and we chose to conduct the satisfaction survey of the patients on the day of discharge and 30 days after discharge.Suggestions for improving compliance include the use of a dedicated wards, specific personnel, effective education and training, and regular inspections . The lacNo serious complications were observed in this study. However, due to our relatively small trial size, which limited our ability to judge the frequency of rare but potentially serious events. PONV is still the main problem affecting the early postoperative feeding and activity of patients. General anesthesia combined with TAP block and multimodal postoperative analgesia pathway could reduce the use of opioids, meanwhile reduce the occurrence of PONV . At presWe also noted that the incidence of preoperative hypokalemia was higher in patients with < 60% compliance, it may be attributed to the no prolonged fasting, as well as insulin resistance associated with oral carbohydrate intake, which deserved further research. Several limitations of the current study need to be addressed. First, a randomized controlled trial should be the preferred option, yet now the existing evidence on ERAS protocols; we considered it unethical to conduct a randomized controlled trial. However, the relatively large sample size, the fixed ERAS MDT and the prospective data collection by a dedicated nurse are strengths of this study. Although most data were entered prospectively, analysis was done retrospectively and results should be interpreted as such. Further researches into the ERAS pathway are needed, including using validated international classification systems such as Clavien-Dindo, and evaluation of additional patient-related outcomes such as patient experience of the process and longer-term consequences. In addition, the predictive nomogram model without a subsequent validation study, it cannot be extrapolated. And the validation and optimization of current model needed to be performed in future study.In summary, better compliance to the ERAS protocols modified for gynecological surgery is crucial to postoperative quality of recovery after benign hysterectomy, decrease the overall complication rate and improve patient experience without a significant increase in readmission and mortality. In this study in particular, minimally invasive surgery, PONV prophylaxis, early mobilization, early oral intake, and early removal of urinary drainage were associated with a lower complication rate after benign hysterectomy.Additional file 1. ERAS protocol used in our department.Additional file 2. QoR-15 questionnaire used in the study.Additional file 3. Definition of postoperative complications in the study.


SERPINA1 Z allele causes α1-antitrypsin deficiency, a rare condition that can cause lung and liver disease. However, the effects of Z allele heterozygosity on nonrespiratory phenotypes, and on lung function in the general population, remain unclear.Homozygosity for the We conducted a large, population-based study to determine Z allele effects on >2400 phenotypes in the UK Biobank (N=303 353).−68), and with other nonrespiratory phenotypes including increased risk of gall bladder disease, reduced risk of heart disease and lower blood pressure, reduced risk of osteoarthritis and reduced bone mineral density, increased risk of headache and enlarged prostate, as well as with blood biomarkers of liver function. Heterozygosity was associated with higher height-adjusted forced expiratory volume in 1 s (FEV1)  and FEV1/forced vital capacity  in nonsmokers, whereas in smokers, this protective effect was abolished. Furthermore, we show for the first time that sex modifies the association of the Z allele on lung function.Z allele heterozygosity was strongly associated with increased height  as well as a variety of nonrespiratory diseases and traits, including liver-, heart- and bone-related conditionsHeterozygosity for the https://bit.ly/37XC0Yg SERPINA1 Z allele (rs28929474(T)) is the commonest cause of severe α1-antitrypsin deficiency (AATD) and is a well-established genetic risk factor for lung diseases such as chronic obstructive pulmonary disease (COPD). However, the health consequences of heterozygosity for the Z allele are not as well-understood ; whereas if cys>0.8, then eGFR=133*((cys/0.8)**−1.328)*(0.996**age)*[0.932 if female].For continuous traits, linear regression models adjusted for sex, age, ageIn ALSPAC, the association of Z allele heterozygosity with height and lung function z-scores were examined using linear regression models including age and gender .1/FVC were generated in UK Biobank using Plink version 1.90b3. These were based on 60 730 autosomal variants with minor allele frequency >1% and p<0.05 (for the association with FEV1/FVC in UK Biobank), and linkage disequilibrium pruned to r2<0.1 in 250-kb windows. Variants were weighted by FEV1/FVC β-coefficients from the SpiroMeta consortium cohorts [Polygenic risk scores for lower FEV cohorts .table S1), consistent with reduced survival.Amongst 411 002 unrelated, European UK Biobank participants with full sex, age, height, smoking status and genotyping data, 16 199 (3.94%) were heterozygous and 129 (0.0314%) were homozygous for the Z allele. There were fewer ZZ homozygotes than expected under Hardy–Weinberg equilibrium in all samples (p=0.004), ever-smokers (p=0.003), individuals >60 years of age (p=0.007) and females (p=0.007) (tables S2 and S3). Standing height, AATD and various respiratory phenotypes  were strongly associated with the Z allele under either genetic model. However, we also detected strong associations between Z allele heterozygosity and a number of other phenotypes, including increased risk of gall stones , gall bladder removal  and bile duct disease ; cardiovascular phenotypes such as lower risk of heart disease  and family history of heart disease, and lower blood pressure (p=1.02×10−10); musculoskeletal phenotypes such as a higher grip strength (p=5.67×10−7), lower risk of osteoarthritis, particularly of knee or hip , lower bone mineral density (p=1.40×10−5) and higher risk of osteoporosis ; and higher risk of headache  and enlarged prostate . The PheWAS also included blood counts for a variety of different cell types, showing reduced reticulocytes in individuals heterozygous for the Z allele. Z allele homozygosity was associated with increased haemoglobin (p=7.17×10−10), haematocrit (p=6.17×10−8) and red blood cells (p=5.91×10−5), as well as optic neuritis  and pancreatitis .To explore the effect of Z allele heterozygosity and homozygosity on a broad range of phenotypes, we performed two PheWASs. The phenotypes most strongly associated with Z allele heterozygosity and Z allele homozygosity (FDR <0.01) are shown in −1, p=2.95×10−25), higher albumin , higher alkaline phosphatase (ALP) , higher aspartate aminotransferase (AST)  and higher direct bilirubin . There were also strong associations with calcium , cystatin C , C-reactive protein  and insulin-like growth factor (IGF)-1 . Heterozygotes exhibited higher sex hormone-binding globulin (SHBG)  and testosterone . The association with higher testosterone was driven by heterozygosity in males  but not females .To extend these analyses to traits not previously available for the PheWAS, we investigated the effect of Z allele heterozygosity and homozygosity on 30 key blood biochemistry markers in UK Biobank data . HeterozSERPINA1 Z allele with lung function traits in unrelated, European UK Biobank participants with full sex, age, height, smoking status, lung function and genotyping data  but no association with FEV1/FVC or FVC compared to wild type  and increased FEV1/FVC  in never-smokers, but not in ever-smokers . Statistical tests of Z allele*ever-smoking interactions showed interactions for FEV1 (p=0.022) and FEV1/FVC (p=1.06×10−4) (table S4). Furthermore, heterozygous ever-smokers exhibited a small increased risk of COPD  and COPD GOLD 1–4 ), whereas heterozygous never-smokers did not (We tested association of heterozygosity for the ing data . Individild type . However-smokers . The FEV did not .−68)  and FVC  but not FEV1/FVC. These results suggest that carrying one copy of the Z allele confers an advantage primarily (but not entirely) driven by increased height. To investigate the developmental stage at which Z allele heterozygosity influences height, we tested association with height in the ALPSAC cohort at ages 8, 15 and 24 years. The findings were consistent with Z allele heterozygosity influencing height from adolescence and, at all the ages, directions of effect on height and lung function were consistent with those in UK Biobank (table S5).Heterozygosity for the Z allele was strongly associated with height   and, whe1/FVC in females  but not in males   (tables S4 and S6).We also assessed whether sex can modify the effect of the Z allele on lung function or height and found that heterozygosity for the Z allele was associated with higher height-adjusted FEV1 , FEV1/FVC  and FVC  compared to wild type and heterozygous individuals, and greater risk of COPD  and COPD GOLD 1–4 ) (1/FVC <0.7) and four out of 15 homozygous ever-smokers aged >60 years did not have spirometrically defined COPD. We generated polygenic risk scores for lower FEV1/FVC by weighting variants according to β-coefficients in the SpiroMeta consortium cohorts [In contrast to the lung function-raising effects of carrying one copy of the Z allele, homozygosity for the Z allele has been widely reported to reduce lung function and increase risk of COPD. Similarly, UK Biobank individuals homozygous for the Z allele had significantly lower FEV×10−12)) . The ass×10−12)) . Given t cohorts . The 58 We note that only 20 out of 141 individuals who were genotyped ZZ homozygotes in the full UK Biobank cohort had a recorded ICD-10 coding for AATD (E880) in HES. As this could indicate miscoding rather than misdiagnosis, we investigated further in a subset of 65 homozygotes with linked primary care data. Of these 65 ZZ homozygotes, 15 had primary care (Read) codes or ICD-10 codes for AATD and 50 did not. Of the group without AATD codes, four had Read codes or ICD-10 codes for bronchiectasis and an additional six had primary care (Read) codes or ICD-10 codes for COPD, indicating potential misdiagnosis of AATD.SERPINA1 from a PheWAS of UK Biobank, and present definitive evidence that heterozygosity for the Z allele is associated with greater FEV1 and FEV1/FVC only in nonsmokers. Our findings have implications for individuals heterozygous for the SERPINA1 Z allele, and more generally for the study of rare variants and gene–environment interactions.We describe novel phenotypic associations of heterozygosity for the Z allele of SERPINA1 Z allele revealed potentially important associations with nonrespiratory traits and diseases. Consistent with previous reports [Our PheWAS examining the consequences of carrying one copy of the  reports , we deteWe detected strong associations between heterozygosity for the Z allele and increased risk of gall stones and gall bladder removal. This is consistent with recent reports that the Z allele may be a risk factor for developing gallstone disease , 22. The1-antitrypsin (AAT) can inhibit receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation and bone resorption [Heterozygosity for the Z allele was also associated with musculoskeletal phenotypes such as reduced risk of osteoarthritis, increased risk of osteoporosis and lower bone mineral density. Calcium was increased in heterozygotes but, as albumin was also increased, free calcium may not be different in this group. A recent study showed that αsorption . As hetesorption , 27.Heterozygotes for the Z allele also had reduced risk of heart disease (even when adjusted for height) and lower blood pressure, as well as increased risk of headache, consistent with one previous report suggesting cluster headaches may be more frequent in heterozygotes for the Z allele . There wHomozygosity for the Z allele was strongly associated with self-reported/HES-recorded AATD and respiratory traits but also haemoglobin concentration and haematocrit percentage, which may be driven by the associations with respiratory disease. Homozygosity was also associated with markedly higher risk of optic neuritis and pancreatitis. These associations should be interpreted with care, especially the association with optic neuritis, as small case numbers may have resulted in poor estimation of effect sizes.While homozygosity for the Z allele is an established cause of respiratory disease, the effect of heterozygosity on the respiratory health of general populations has been less clear. Our study provides definitive data on the effects of heterozygosity for the Z allele on respiratory traits, showing that heterozygosity is associated with greater lung function in never-smokers (even after height adjustment) but not in smokers. Our results contrast with previous studies –11 that SERPINA1 gene encodes AAT, a glycoprotein with an important role in inhibiting proteases, such as neutrophil elastase, which are secreted during inflammation and cause collateral tissue damage. The Z allele produces AAT with reduced antiprotease activity and a propensity to form polymers, which accumulate in liver, thereby reducing the circulating levels of AAT. The polymers themselves have also been shown to be a proinflammatory stimulus in lung tissue [The biological mechanism underlying the association between heterozygosity for the Z allele and increased lung function in nonsmokers is not clear. The g tissue , which fg tissue . One migg tissue , 31 and g tissue  and protg tissue . Cigaretg tissue , suggestSERPINA1 Z allele heterozygosity on FEV1/FVC. Women heterozygous for the Z allele exhibited greater FEV1/FVC compared to wild-type, whereas men heterozygous for the Z allele showed no significant difference to wild type. Previous studies have shown that individuals with the fastest decline in lung function amongst AATD patients are more likely to be male [We also report a novel interaction between sex and the  be male . Howeverakanishiet al. [Finally, we confirm that homozygosity for the Z allele is associated with reduced lung function and increased risk of COPD, and that these effects are exacerbated by smoking. These results are consistent with a recently published study in UK Biobank focused on homozygosity for the Z allele . Also coiet al. , we showiet al. , 36), anSERPINA1 Z allele. Our findings suggest that while individuals who are heterozygous for the Z allele may not exhibit symptoms of AATD, they may be more susceptible to smoking-induced lung disease and have altered risk of other nonrespiratory conditions.In conclusion, we have demonstrated that heterozygosity for the Z allele is associated with previously unrecognised nonrespiratory phenotypes. Further studies will be required to confirm these findings in independent populations; produce accurate effect estimates; and understand the causal pathways involved, and the potential role of genetic and environmental modifiers. We have also shown that heterozygosity for the Z allele is associated with greater lung function in nonsmokers. Our results demonstrate that large sample sizes are required to study the effects of rare variants, particularly when these effects depend on an environmental stimulus. The opposing directions of effect of Z allele heterozygosity and homozygosity on lung function demonstrate that the additive genetic model is not always appropriate for genetic studies of rarer variants such as the 10.1183/23120541.00049-2021.Supp1Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author.00049-2021.SUPPLEMENTSupplementary material 00049-2021.TableS1Table S1 00049-2021.TableS2Table S2 00049-2021.TableS3Table S3 00049-2021.TableS4Table S4 00049-2021.TableS5Table S5 00049-2021.TableS6Table S6


Pegylated liposomal doxorubicin (PLD) is an improved formulation of doxorubicin with comparable effectiveness but significantly lower cardiotoxicity than conventional anthracycline. This study aimed to evaluate the real-world effectiveness and safety of PLD versus epirubicin as neoadjuvant or adjuvant treatment for breast cancer.Clinical data of invasive breast cancer patients who received neoadjuvant or adjuvant chemotherapy with PLD or epirubicin were retrospectively collected. Propensity score matching (PSM) was performed to reduce the risk of selection bias. The molecular typing of these patients included Luminal A, Luminal B, HER2-positive, and basal-like/triple-negative. The primary outcome was pathological complete response (pCR) rate for neoadjuvant chemotherapy and 3-year disease-free survival (DFS) rate for adjuvant chemotherapy. Noninferiority was suggested if the lower limit of the 95% CI for the 3-year DFS rate difference was greater than − 10%. The secondary outcome was adverse reactions.n = 274; adjuvant, n = 939). pCR (ypT0/Tis ypN0) rates of patients who received neoadjuvant chemotherapy were 11.6% for the PLD group and 7.0% for the epirubicin group, but the difference was not statistically significant (P = 0.4578). The 3-year DFS rate of patients who received adjuvant chemotherapy was 94.9%  for the PLD group and 95.4%  for the epirubicin group (P = 0.5684). Rate difference between the two groups and its 95% CI was - 0.55 . The lower limit of the 95% CI was − 5.0% > − 10.0%, suggesting that PLD is not be inferior to epirubicin in adjuvant chemotherapy for breast cancer. The incidences of myelosuppression, decreased appetite, alopecia, gastrointestinal reactions, and cardiotoxicity were lower in the PLD group than in the epirubicin group, while the incidence of nausea was higher in the PLD group.A total of 1213 patients were included (neoadjuvant, In the neoadjuvant and adjuvant treatment of breast cancer, effectiveness is similar but toxicities are different between the PLD-containing regimen and epirubicin-containing regimen. Therefore, further study is warranted to explore PLD-based neoadjuvant and adjuvant chemotherapy for breast cancer. Breast cancer is the most common malignancy in women worldwide. According to the 2018 statistics of the International Agency for Research on Cancer (IARC) of the World Health Organization, there were 2.08 million new cases of breast cancer and 620,000 breast cancer-related deaths in the world, which accounted for 24.2 and 15% of all malignancies and malignancy-related deaths in women, respectively .Anthracycline-based chemotherapy is a common neoadjuvant and adjuvant therapy for breast cancer patients. The recommended anthracycline drugs include doxorubicin and epirubicin . Anthrac2, and the breast pCR rate was 18.8%  with no significant decrease in LVEF [Pegylated liposomal doxorubicin (PLD) is a liposomal formulation of doxorubicin with comparable effectiveness but markedly lower cardiotoxicity than conventional anthracycline , thus al in LVEF , 15. Ano in LVEF .This real-world study aimed to compare the effectiveness and safety of PLD to epirubicin as neoadjuvant or adjuvant treatment for breast cancer patients.The medical records of breast cancer patients who received PLD  or epirubicin-based neoadjuvant (January 2014 to January 2018) or adjuvant treatment (June 2014 to June 2016) were retrospectively collected. Inclusion criteria: 18–70 years old; female; histologically confirmed invasive breast cancer; received PLD or epirubicin neoadjuvant chemotherapy or adjuvant chemotherapy; LVEF ≥50%. Exclusion criteria: occult breast cancer patients; used two or more anthracyclines during neoadjuvant or adjuvant chemotherapy; previously received other chemotherapy regimens. After screening, the patients were divided into the neoadjuvant chemotherapy group and adjuvant chemotherapy group according to their treatment stage and then into the PLD group and epirubicin group according to the drug regimen.The study was approved by the Ethics Research Committee of Tianjin Cancer Hospital.Trial Registration: ClinicalTrials.gov, Identifier: NCT03983096.The expression statuses of ER, PR, HER2, and Ki67 were detected by immunohistochemical staining to determine the molecular subtyping. ER and PR were considered positive when more than 1% of the tumor cells exhibited positive staining. For HER2 staining, a score of 3+ was considered positive; a specimen with a score of 2+ was tested by fluorescence in situ hybridization analysis. The standard threshold value of Ki67 was 20%. If ≥20%, it was considered to be high Ki67 expression, otherwise low Ki67 expression. In 2013, the St. Gallen International Breast Cancer Conference defined the molecular classification of breast cancer . When ERCardiotoxicity was defined as abnormal results of cardiac function in clinical evaluation, including decreased ventricular ejection fraction , congestive heart failure, arrhythmia, etc. . CardiotThe primary outcome for neoadjuvant chemotherapy was the total pathological complete response (pCR) rate , which was defined as the absence of residual invasive cancer cells or only carcinoma in situ in the primary and metastatic lymph nodes after surgery. The primary outcome for adjuvant chemotherapy was the 3-year disease-free survival (DFS) rate. DFS referred to the time from the first postoperative chemotherapy to recurrence, metastasis, or death. The secondary outcome was the incidence of adverse reactions.When the baseline characteristics of the eligible patients were balanced, the eligible case data were used for statistical analysis. Otherwise, propensity score-matching (PSM) was conducted to reduce the selection bias between the PLD and epirubicin groups. The variables included in PSM were age, lymph node metastasis, tumor size, and molecular typing.All statistical tests were two-sided with a significance level of α = 0.05. The 3-year DFS rate of adjuvant chemotherapy was compared between the two groups using the chi-square test or Fisher’s exact test. The 95% confidence interval (CI) for the 3-year DFS rate difference was calculated. For exploratory purposes, a noninferiority test was performed, with the noninferiority margin set at − 10%. Noninferiority was suggested if the lower limit of the 95% CI for the 3-year DFS rate difference was greater than − 10% . The pCR rate of neoadjuvant chemotherapy was also compared between the two groups using the chi-square test or Fisher’s exact test. According to the conditions of the patients included, this study conducted subgroup analyses of the main study outcomes for the neoadjuvant chemotherapy and adjuvant chemotherapy population. Factors such as menopausal status , tumor size (T1 or T2), lymph node metastasis , clinical stage (II or IIIA), ER status (positive or negative), PR status (positive or negative), HER2 status (positive or negative), and Ki-67 expression level (< 20% or ≥ 20%) were considered for the subgroup analyses.For safety analysis, the number and incidence of adverse reactions in the PLD group and epirubicin group were counted.The clinical data of 1309 breast cancer patients who were diagnosed and treated in seven hospitals in China between January 2014 and January 2018 were retrospectively reviewed. A total of 1213 patients met the selection criteria , PLD or epirubicin combined with C followed by a taxane (T), T followed by PLD or epirubicin combined with C, PLD or epirubicin combined with T, PLD or epirubicin combined with T and C, and PLD or epirubicin combined with C and 5-fluorouracil Table . The dosP = 0.0353), tumor size (P = 0.0452), and lymph node metastasis (P = 0.0109), were not evenly distributed across the PLD and epirubicin groups before PSM. After one-to-one PSM, there were 43 patients in each group, and the baseline characteristics of the two groups were balanced  had evaluable effectiveness (79 patients discontinued neoadjuvant treatment due to unknown reasons and lacked the results of pCR), including 65  patients who received PLD-containing regimen, and 130  patients who received epirubicin-containing regimen. The baseline characteristics of the patients, including age < 35 (P = 0.0262) and lymph node metastasis (P = 0.0046), were not evenly distributed between the two groups before PSM. After PSM (1:2), there were 201 patients in the PLD group and 402 patients in the epirubicin group, and the baseline characteristics were balanced between the two groups  patients received a PLD-containing regimen, and 647 (68.9%) patients received an epirubicin-containing regimen. The baseline characteristics of the patients, namely age < 35 (1.1% patiP = 0.3270). Breast pCR  was also higher in the PLD group  than in the epirubicin group , but the difference was not statistically significant (P = 0.1173)  than in the epirubicin group , but the difference was not statistically significant  and bpCR  were also comparable between the PLD group and epirubicin group  could not be performed.P = 0.6516)  and epirubicin groups  (P = 0.5684)  and epirubicin groups  (84) Fig. B. The raAfter PSM, the 3-year DFS rate was analyzed by subgroups according to the menopause status , tumor size (T1 or T2), lymph node metastasis , clinical stage (II or IIIA), ER (positive or negative), PR (positive or negative), HER-2 (positive or negative), Ki-67 expression (< 20% or ≥ 20%), and histological grade (grade II or III). 3-year DFS rate was higher in premenopausal, T2, N2, stage II, Ki-67 ≥ 20% patients in the PLD group than in the epirubicin group  who received neoadjuvant chemotherapy or adjuvant chemotherapy were included in the safety analysis. According to the medical records of the patients, the incidence of adverse reactions was lower in the PLD group (15.2%) than in the epirubicin group (18.1%). The common adverse reactions were myelosuppression, decreased appetite, cardiotoxicity, and gastrointestinal reactions  as those in the epirubicin group [pCR (ypT0/is or ypT0/is ypN0) is a standard effectiveness outcome of neoadjuvant therapy for breast cancer. Pooled analysis showed that patients who achieved pCR have improved survival , 20. Prein group . Yao et in group .P < 0.0001) and 5-year OS rates  of the 2391 breast cancer patients receiving adjuvant therapy [P = 0.5684), and the lower limit of 95% CI of the rate difference between the two groups was − 5.0% > − 10.0%, which indicated that PLD was not inferior to epirubicin as adjuvant therapy for breast cancer. Nevertheless, the follow-up was only 3 years. The patients are still being followed, and the results will be updated.Adjuvant therapy is an important treatment for early breast cancer patients as it significantly reduces the risk of recurrence and improves patient survival –32. Anth therapy . In addi therapy . Another therapy . SimilarThere is increasing evidence that PLD can significantly reduce the risk of cardiotoxicity compared with other anthracyclines , 26, 38.In this study, we used a matched case-control design with stringent matching criteria to compare the effectiveness and safety of PLD vs. epirubicin as neoadjuvant or adjuvant chemotherapy in breast cancer patients who received the treatment within the same period. Patients who received adjuvant chemotherapy were followed up for at least 3 years to obtain their long-term survival benefit data. However, the long-term benefit of PLD is still unclear since the diagnosis and treatment data analyzed in this study were collected from only seven hospitals.


In neurodegenerative diseases including Alzheimer’s, Parkinson’s and prion diseases, astrocytes acquire disease-associated reactive phenotypes. With growing appreciation of their role in chronic neurodegeneration, the questions whether astrocytes lose their ability to perform homeostatic functions in the reactive states and whether the reactive phenotypes are neurotoxic or neuroprotective remain unsettled. The current work examined region-specific changes in expression of genes, which report on astrocyte physiological functions and their reactive states, in C57Black/6J mice challenged with four prion strains via two inoculation routes. Unexpectedly, strong reverse correlation between the incubation time to the diseases and the degree of astrocyte activation along with disturbance in functional pathways was observed. The animal groups with the most severe astrocyte response and degree of activation showed the most rapid disease progression. The degree of activation tightly intertwined with the global transformation of the homeostatic state, characterized by disturbances in multiple gene sets responsible for normal physiological functions producing a neurotoxic, reactive phenotype as a net result. The neurotoxic reactive phenotype exhibited a universal gene signature regardless of the prion strain. The current work suggests that the degree of astrocyte activation along with the disturbance in their physiological pathways contribute to the faster progression of disease and perhaps even drive prion pathogenesis.The online version contains supplementary material available at 10.1186/s40478-021-01192-9. Transformation of astrocytes into reactive states is recognized as one of the major hallmarks of neurodegenerative diseases including Alzheimer’s, Parkinson’s, Amyotrophic Lateral Sclerosis and prion diseases , 2. The Several recent studies suggested that astrocytes are intimately involved in prion disease pathogenesis, yet their role remains poorly defined –16. In pThe current study examined region-specific changes in astrocyte function and their reactive phenotype by monitoring the expression of astrocyte-specific genes along with markers of astrocyte reactive states in animals challenged with four prion stains via two different routes. One can expect the most severe changes in the degree of astrocyte activation and intensity of their response occur in animal groups with long incubation times to disease due to longer exposure to altered brain homeostasis. Instead, strong reverse correlation between the degree of astrocyte activation and the incubation time to terminal diseases was observed. Reactive states associated with prion diseases involved global transformation of the physiological functions, characterized by disturbance in multiple physiological pathways. The current work illustrated that the degree of astrocyte activation along with loss of normal homeostatic functions is predictive of the incubation time to the diseases, and suggests that these changes perhaps even drive prion disease progression.2 asphyxiation and their brains were immediately extracted. The brains were kept ice-cold for prompt dissection or preserved in 10% buffered formalin (MilliporeSigma) for histopathology.Using isoflurane anesthesia, 6-week-old C57Black/6J female and male mice were inoculated with 10% or 1% brain homogenates, as indicated, prepared in PBS (pH 7.4) using terminally ill 22L-, ME7-, RML-, or SSLOW-infected mice. Brain materials from the 5th passage of SSLOW was used for inoculations , 19. Inohttp://mouse.brain-map.org/static/atlas) was used as a reference. The hypothalami (HTh), as well as thalami (Th), hippocampi (Hp) and cortices (Ctx) were collected into RNase-free, sterile tubes, frozen in liquid nitrogen and stored at −80˚C until the RNA isolation with Aurum Total RNA Mini Kit , as described [Extracted ice-cold brains were dissected using a rodent brain slicer matrix . A 2 mm central coronal section of each brain was used to collect individual regions. Allen Brain Atlas digital portal (escribed .www.brainrnaseq.org and https://www.networkglia.eu/en/astrocyte) were used for selecting 275 genes that under normal conditions, express primarily in astrocytes and were assigned to functional pathways. In addition, 47 genes, reporting on reactive phenotypes, including A1-, A2- and pan-specific makers along with other markers of reactive astrocytes were used for the panel, as well as 8 microglia-, 10 neuron-, and 2 oligodendrocyte-specific genes. The addition of 10 housekeeping genes completed the panel bringing total number of genes to 352. These genes were assigned to 23 gene sets to allow for an advanced analysis of their group changes.To design custom-based nCounter Mouse Astrocyte Panel . For agglomerative clusters and heat maps, genes with less than 10% of samples above 20 counts were excluded. Z-score transformation was performed for genes. Clustering was done using Euclidian distance and the linkage method was Average.n = 3) against the control normal i.p. female (F) group (n = 6). Only genes with p < 0.05 and linear fold change ≥  ± 1.2 were counted. Differential expression analysis for Fig. p < 0.05 and linear fold change ≥  ± 1.2 were counted. Advanced Analysis was performed for each brain region separately. Undirected global significance scores obtained as a result of gene set analysis (GSA scores) were added up to produce a combined GSA score  for plots on Additional file For Fig. Formalin-fixed brains were treated for 1 h with 95% formic acid to deactivate prion infectivity before being embedded in paraffin. Subsequently, 4 µm brain sections produced using a Leica RM2235 microtome were mounted on slides and processed for immunohistochemistry. To expose epitopes, slides were subjected to 20 min of hydrated autoclaving at 121 °C in Antigen Retriever citrate buffer, pH 6.0 . Chicken polyclonal anti-GFAP  was used to stain for astrocytes. Detection was performed with a DAB Substrate Kit (BD Biosciences).For co-immunofluorescence, rabbit polyclonal anti-AQP4 antibody  and chicken polyclonal anti-VIM  were used in combination with chicken polyclonal anti-GFAP  or rabbit anti-GFAP clone D1F4Q , respectively. The secondary antibodies were goat anti–rabbit or anti-chicken IgG conjugated with Alexa Fluor 546 for red color or Alexa Fluor 488 for green color (Thermo Fisher Scientific). Images were collected with an inverted microscope (Nikon Eclipse TE2000-U) equipped with an illumination system X-cite 120  and a cooled 12-bit CoolSnap HQ CCD camera . Fiji ImageJ software was used for image analysis.t test (****p < 0.0001).AQP4 distribution was measured on the images taken with 20× objective. 50 pixel long, linear profiles across cortical microvessels were exported to Microsoft Excel. The lines were drawn avoiding cell bodies, positioning the vessel in the middle. Measurements were performed on the brains of 4 infected animals and 3 control animals . The minimum value of each profile was subtracted as a background value. Non-perivascular AQP4 signal was reported as an average intensity of the first and last 15 pixels of each profile. Maximum intensity of the 20 pixels in the middle of each plot represented the perivascular AQP4 signal. The ratio of perivascular AQP4 signal to the non-perivascular signal for each brain was plotted, and the statistical significance of the difference between infected and normal brains was estimated by an unpaired 2 and p-values were calculated using simple linear regression analysis performed with GraphPad Prism 9.0.0. In Fig. p < 0.0001).In Figs. www.brainrnaseq.org and https://www.networkglia.eu/en/astrocyte)  and male (n = 3) mice were assessed by the astrocytic panel  between the total number of DEGs and incubation time to disease emerged.For examining the changes associated with prion diseases, C57Black/6J mice were inoculated intraperitoneally (i.p.) with 22L, ME7 or SSLOW mouse-adapted prion strains and analyzed at the terminal stage of the disease  with SSLOW, 22L, RML or ME7; animals challenged via i.p. with RML; and an additional SSLOW group inoculated via i.p. , the strength of the relationship was even higher for the i.c. groups (R2 = 0.84), but weaker for the i.p groups (R2 = 0.64),  and Th (i.c. group) showed weak correlations  to the incubation time to disease. They included pathways reporting on the astrocytes reactive states or involved in regulating astrocyte reactivity , as well as multiple pathways reporting on astrocyte function   between the gene sets reporting on the degree of activation and the dysregulation in physiological pathways , suggesting that in reactive astrocytes, both sets change in parallel  and neuroligin 1 (Nlgn1), the cell surface proteins that are required for neurotransmission by mediating formation and maintenance of synapses [Cdh10), which is involved in maintenance of excitatory and inhibitory synaptic structures [Gpc4, Gpc5), which are important for functional synapse formation [Both sets of genes, involved in neuroprotection and neurotoxicity, were among the groups with a strong relationship to the incubation time Fig. . In factsynapses , 36. Sevructures , and glyormation . While tIn a healthy brain, astrocytes are responsible for a number of important physiological functions including support of neuronal growth, modulation of neurotransmission, formation and maintenance of synapses, regulation of blood flow, supplying energy and providing metabolic support to neurons, maintaining the blood–brain-barrier and more –41. In n2 = 0.84), lower for the combined i.c. and i.p. groups (R2 = 0.69), and the lowest, yet still robust and highly significant, for the i.p. groups (R2 = 0.62). Strain-specific differences in pathogenic steps that preceded neuroinvasion such as interaction of prions with peripheral immune cells, replication in secondary lymphoid organs and trafficking to the CNS [For addressing the above questions, the current work examined animals challenged with four prion strains via two inoculation routes using a gene panel that monitored expression of astrocyte-specific genes. Under the circumstances where astrocytes simply respond to changes in the CNS environment, one should expect more severe changes in groups with longer incubation times, due to longer exposure to altered brain homeostasis along with a more pronounced impact of natural aging. Indeed, in normal aged animals, dysregulation in astrocytic gene expression was found to be reminiscent of those observed in neurodegenerative diseases –47. More the CNS –33, wereAs illustrated by a region-specific transcriptome profile Fig. , astrocyThe four prion strains used in the current work exhibit different cell-specific tropism. 22L is mainly associated with astrocytes, ME7 is primarily found in neurons, SSLOW is colocalized with microglia and considerably less with astrocytes, whereas cell association of RML depends on brain region, showing either neuron- or astrocyte-specific localization , 24. RegWhat normal functions of astrocytes are disturbed in prion diseases? The fold changes for the individual functional genes, including the top 40 functional genes . These results are in agreement with previous studies [Changes in functional states of astrocytes occurred in parallel with the transformation in their reactive state. According to the Barres hypothesis, astrocytes can polarize and acquire either neurotoxic (A1) or neuroprotective (A2) phenotypes, which exhibit distinct transcriptome characteristics and, presumably, opposing effects on neuronal survival , 59. In  studies , 19, 44  studies . The curIn the current work, mRNA expression was analyzed in bulk tissues opening possibilities that changes in gene expression were attributed not only to astrocytes, but also to other cell types. Indeed, despite the fact that pan-, A1- and A2-markers have been used widely to describe the reactive state of astrocytes, the majority of these markers along with genes responsible for neuroinflammation are known to be expressed by multiple cell types . The pos+VIM−, GFAP+VIM+ and GFAP+ astrocytes with low levels of VIM expression. We do not know, whether heterogeneity in VIM expression levels represent different degrees of astrocyte activation, different sub-populations of disease-associated astrocytes, normal vs disease-associated subpopulations or all of the above. It will be interesting to examine in future studies whether reactive astrocytes associated with prion diseases are shared with other neurodegenerative diseases or prion-specific.Previous studies that employed single-cell RNA sequencing established that astrocytes not only display region-specific homeostatic signatures, but also exhibit subregional heterogeneity defined by developmental patterns –22, 60. Nrxn1, Nlgn1, Cdh10, Gpc4, Gpc5) were found to be downregulated. While some neuroprotective pathways might be upregulated in response to prions, the net result of disturbances in neuroprotective/neurotoxic pathways, along with the global dysregulation across physiological functions, produced a neurotoxic phenotype. Indeed, several recent studies argue that reactive astrocytes associated with prion diseases are neurotoxic. Primary astrocytes isolated from prion-infected animals had adverse effects on neuronal cultures invoking reduction in spine size and density along with impairment of neuronal growth and synapse integrity [The current work suggests that phenotypic changes in the state of astrocytes contribute to the faster progression of diseases and perhaps even drive prion pathogenesis. Notably, both sets of genes, those involved in neuroprotection and neurotoxic functions, were disturbed in the reactive astrocytes. It appears that in the reactive states, astrocytes upregulate a group of genes associated with neuronal protection, which is in agreement with previous work documenting upregulation of defense pathways against protein misfolding and reactive oxygen species . In parantegrity . Synaptontegrity . Selectintegrity . Moreoventegrity . TogetheSc-astrocyte interaction is important for their activation, and to what molecular features of PrPSc, if any, astrocytes respond. Recent studies that employed triple TNF−/−/IL1a−/−/C1qa−/− knockout mice questioned the role of reactive microglia as a driver of the neurotoxic phenotypes in astrocytes [Sc [Sc over a certain threshold, and the kinetics in GFAP overexpression followed very closely the kinetics of PrPSc accumulation [Sc directly and, in response, upregulate expression of chemokine genes triggering microglia migration [Sc with a pro-inflammatory response, where the degree of the response was found to be dictated by the sialylation status of N-linked glycans on the surface of PrPSc [Sc sialylation varies among prion strains [Sc sialylation pattern accompanied the shortening of the incubation times to the disease during serial adaptation of a prion strain to a new species, suggesting that the PrPSc sialylation pattern dictate glial activation and disease progression [What factors dictate the incubation time to the diseases? Physical properties of prions including strain-specific replication rate, conformational stability and size of protein aggregates were examined in the past in attempt to establish a link between strain-specific characteristics and disease progression –64. The trocytes . Eliminatrocytes . Moreovetrocytes . In neurytes [Sc . In prevytes [Sc . In micemulation . Furtherigration . Microglof PrPSc . Interes strains , 70. Notgression , 71. In Additional file 1. Table S1: Composition of nCounter Astrocyte panel. Gray font color and asterisks indicate probes producing low counts.Additional file 2. Table S2: List of animal groups analyzed using the Astrocyte panel.Additional file 3. Table S3: Normalized counts for all individual animals listed in Table S2, reported for four brain regions.Additional file 4. Table S4 Linear fold change for each gene, reported as a ratio between each sample group and normal i.p. female (F) as a control group. The table includes data for four brain regions, presented as full lists of genes and as lists of changed genes .Additional file 5. Table S5: List of gene sets in Astrocyte panel. Correlations  between individual gene sets and incubation time to terminal disease calculated separately for four brain regions for i.c. and i.p. groups.Additional file 6. Table S6: Top 40 DEGs in pathways that report on astrocyte function. Fold change along with p-values of the top 40 DEGs expressed in thalamus of all animal groups.Additional file 7.: Figure S1. GFAP in normal and infected brains. (a) GFAP immunoreactivity in cortex, hippocampus and thalamus of normal mice and of mice inoculated i.p. with 22L or SSLOW. Scale bar 100 μm. (b) Normalized GFAP counts detected by NanoString in cortex, hippocampus and thalamus of normal mice and of mice inoculated i.p. with 22L or SSLOW (n = 3). Figure S2. Linear regression for the combined GSA scores in four brain regions. Inverse correlations between the combined GSA scores and the time to terminal prion disease in cortex (Ctx), hippocampus (Hp), thalamus (Th) and hypothalamus (HTh) of i.p. and i.c. sample groups.  Figure S3. DEGs in neurotoxicity and neuroprotection gene sets in four prion strains. (a) Log2 fold changes in neurotoxicity and neuroprotection gene sets in thalamus of male mice i.c.-inoculated with ME7, 22L, RML, or SSLOW . (b) Correlation between undirected global significance scores of neurotoxicity and neuroprotection gene sets calculated for thalamus of female and male groups inoculated via i.p. and i.c. routes.Figure S4. DEGs in neurotoxicity and neuroprotection gene sets in four brain regions. Log2 fold changes in neurotoxicity and neuroprotection gene sets in cortex (Ctx), hippocampus (Hp), thalamus (Th) and hypothalamus (HTh) of female mice i.c.-inoculated with SSLOW .