Background: People with a Fontan circulation usually have moderately impaired exercise performance, although a subset have high physical performance (“Super-Fontan”), which may represent a low-risk phenotype.Methods: People with a “Super-Fontan” phenotype were defined as achieving normal exercise performance [≥80% predicted peak oxygen uptake (VO2) and work rate] during cardiopulmonary exercise testing (CPET) and were identified from the Australian and New Zealand Fontan Registry. A Fontan control group that included people with impaired exercise performance (<80% predicted VO2 or work rate) was also identified based on a 1:3 allocation ratio. A subset of participants were prospectively recruited and completed a series of physical activity, exercise self-efficacy, and health-related quality of life questionnaires.Results: Sixty CPETs  were included. A subset  completed a series of questionnaires. Average age was 29 ± 8 years; 48% were males. Exercise capacity reflected by percent predicted VO2 was 67 ± 17% in the entire cohort. Compared to the “Super-Fontan” phenotype, age at Fontan completion was higher in controls . Only one (7%) person in the “Super-Fontan” group had a dominant right ventricle compared to 15 (33%) controls (p = 0.043). None of those in the “Super-Fontan” group were obese, while almost a quarter (22%) of controls were obese based on body mass index (p = 0.046). Lung function abnormalities were less prevalent in the “Super-Fontan” group . Exercise self-efficacy was greater in the “Super-Fontan” group . Self-reported sports participation and physical activity levels during childhood and early adulthood were higher in the “Super-Fontan” group (p < 0.05). The total average time spent participating in structured sports and physical activity was 4.3 ± 2.6 h/wk in the “Super-Fontan” group compared to 2.0 ± 3.0 h/wk in controls, p = 0.003. There were no differences in self-reported current total physical activity score or health-related quality of life between groups (p ≥ 0.05).Conclusions: The “Super-Fontan” phenotype is associated with a healthy weight, lower age at Fontan completion, better exercise self-efficacy, and higher overall levels of sport and physical activity participation during physical development. Francis Fontan first described the Fontan procedure in 1971 as a surgical method to treat babies born with tricuspid atresia (2) is the primary index of exercise tolerance  and has significant prognostic value in patients with congenital heart disease  results and a “Super-Fontan” phenotype were identified and included in this study. People with impaired exercise capacity were also identified as controls based on a 1:3 allocation. Exercise and work capacity was measured by peak VOl values , 11. Parl values  or a conl values , 12–15. We decided to include work capacity as a criterion as obese patients may have normal exercise capacity but present with limited work capacity and exercise intolerance. Participants were excluded if their CPET was conducted on a treadmill or if results were considered to be submaximal effort defined as a peak respiratory exchange ratio <1.0 . A subseExercise self-efficacy was assessed using the Exercise Self-Efficacy Scale . The totHealth-related quality of life was measured using the PedsQL Adult Quality of Life Inventory Version 4. The items of each question were reversed scored, and linearly transformed in accordance with the scoring guidelines. In addition to the total score, the physical health summary score and psychosocial health summary score were also calculated, with higher scores suggesting better health-related quality of life.2, work rate, and pre-exercise spirometry, CPET parameters including minute ventilation (VE), carbon dioxide production (VCO2), blood pressure, VO2 at the anaerobic threshold, heart rate (HR), and arterial oxygen saturation indicated by pulse oximetry were obtained when available. Predicted maximal oxygen pulse (ml/beat)—a surrogate for stroke volume—was calculated by dividing predicted maximal VO2 by predicted maximal HR (220–age) (2 (L/min) (1) x 40, and breathing reserve was calculated as MVV–peak VE or (MVV–peak VE)/MVV x 100. A mechanical ventilatory limitation to exercise was suggested by a breathing reserve of <15% or <11 L/min (2 (mL/kg/min) x peak systolic blood pressure (mm Hg). Ventilatory inefficiency was suggested by a peak VE/VCO2 ratio of >40. A significant fall in oxygen saturation was considered as a decrease of ≥5%.Center specific CPET protocols were performed on an electronically braked cycle ergometer as part of routine clinical care. In addition to measures of peak VO220–age) . A cardi220–age)  or a red (L/min) , 20. Max11 L/min , 21. Pea1/FVC ratio and FVC were below the lower limit of normal.Spirometry parameters were considered as abnormal if values were below the lower limit of normal calculated from the Global Lung Initiative regression equations . Lung fuTo assess structured sport and physical activity participation across the lifespan, we used a modified version of the Kriska long-term recall physical activity questionnaire . ParticiSelf-reported current levels of physical activity and sedentary time were assessed using the International Physical Activity Questionnaire (IPAQ) Long-Form. The IPAQ scores were not truncated, and metabolic minutes per week (MET-min/week) were calculated in accordance with the IPAQ Scoring Manual. Sedentary activity was reflected by sitting time (minutes) per day.t-test or Mann–Whitney U was used as appropriate to compare differences between the “Super-Fontan” group and the control group. Proportions were compared using Pearson Chi-Square. A p-value of < 0.05 was considered as statistically significant.Statistical analysis was performed using IBM SPSS version 26 software . Data are presented as mean ± standard deviation or number (%) unless specified otherwise. The Shapiro-Wilk test or visual inspection of histograms and Q-Q plots were conducted to assess for normal distribution. An independent Detailed participant demographics and characteristics are shown in 2, and 20 people (33%) were overweight or obese. None who had the “Super-Fontan” phenotype were obese based on BMI compared to 22% in the control group (p = 0.046). The majority (70%) had a total cavopulmonary connection, and the average age at Fontan procedure was 6.4 ± 5.0 years. The age at Fontan procedure was lower in the “Super-Fontan” group compared to the control group . Thirty-seven (62%) had a dominant left ventricle, 16 (27%) had a dominant right ventricle, 3 (5%) had biventricular morphology, and 4 (7%) had indeterminate ventricles. A dominant right ventricle was associated with impaired exercise performance (p = 0.043). One person (7%) in the “Super-Fontan” group had a Fontan conversion from an atriopulmonary connection to an extracardiac conduit type circulation. There were no statistically significant differences in age, sex, BMI, type of Fontan procedure, or patent fenestration between groups .The average body mass index (BMI) was 25.9 kg/mp ≥ 0.05 for all).Twenty-three Fontan participants completed the questionnaires. Of the 23 Fontan study group participants, 10 (43%) were in the “Super-Fontan” group, and 13 (57%) were in the control group. There were no differences between groups in baseline demographics for the subset of study participants who completed the questionnaires (p = 0.02).The average total exercise self-efficacy score was higher in the “Super-Fontan” group compared to the control group . There was also no statistically significant difference between the “Super-Fontan” and control groups in the physical health summary score  or psychosocial health summary score .There was no statistically significant difference in total health-related quality of life score between the “Super-Fontan” group and the control group .Detailed CPET and spirometry results are shown in 2 and work rate for the entire cohort was 67 ± 17% and 72 ± 22%, respectively. There was no statistically significant difference in percent predicted maximum HR between the “Super-Fontan” and control groups . Peak circulatory power, HR reserve (HRR), peak VE, and VO2 at anaerobic threshold (percentage of predicted VO2) were higher in the “Super-Fontan” group . There was no difference in exercise-induced desaturation between groups (p = 0.5).The average percent predicted peak VOn = 11; control, n = 29) where the chronotropic index could be calculated, 7 (18%) participants had values outside the normal range. All participants in the “Super-Fontan” group had a chronotropic index within the normal range. In the control group, 4 (14%) participants had a high chronotropic index, and 3 (10%) had a low chronotropic index suggesting cardiovascular limitation and chronotropic insufficiency as inhibitors to exercise performance, respectively. The “Super-Fontan” group also had a higher percent predicted oxygen pulse compared to the control group . When markers of cardiovascular limitation were combined [low peak oxygen pulse (<80% predicted) or a high chronotropic index], no patient with the “Super-Fontan” phenotype had evidence of a cardiovascular limitation to exercise capacity compared to 62% in the control group (p < 0.001).Of the 40 participants  had baseline spirometry recorded, 16 (43%) had normal spirometry function, 20 (54%) had evidence of ventilatory restriction, and 1 (3%) had a pattern suggestive of mixed defect. The “Super-Fontan” group tended to have higher percent predicted FVC compared to the control group . Lung function abnormalities at rest were associated with impaired exercise performance; 2 (20%) patients in the “Super-Fontan” group had ventilatory or mixed defect compared to 19 (70%) in the control group (p = 0.006).Thirty-seven people with a Fontan circulation  who had a mechanical ventilatory limitation also had evidence of ventilatory restriction at rest.p = 0.04). The average h/wk of sports and physical activity participation during high school and early adulthood was 5.2 ± 4.4 h/wk in the “Super-Fontan” group and 2.1 ± 3.1 h/wk in the control group, p = 0.04. There was no statistically significant difference in sport and physical activity participation during older adulthood. The overall average duration of sport and physical activity participation indexed per week was higher in the “Super-Fontan” group compared to the control group .One of the participants in the control group had an incomplete questionnaire and was excluded from the analysis. Results for the Kriska physical activity questionnaire are shown in p = 0.07).The detailed IPAQ results are shown in Despite an absent subpulmonary ventricle in the Fontan circulation, a subset of the population (“Super-Fontans”) can still achieve normal exercise performance, which is associated with increased levels of physical activity early in life, a healthy weight, and earlier age at Fontan completion.In this study, the age at Fontan completion was lower in those with a “Super-Fontan” phenotype, and the absence of obesity or a dominant right ventricle were associated with normal exercise performance. This contrasts with previous findings that showed no differences between exercise performance Fontan phenotypes with these factors , 7. Our 2 and HRR decreased by 1.5 percentage points and 4.1 beats/min, respectively  limitation to exercise performance denoted by an elevated chronotropic response or low peak oxygen pulse. Of course, the reduced oxygen pulse and elevated chronotropic index can also reflect impaired peripheral muscle oxygen extraction, which is also present in the Fontan circulation  time was lower in the “Super-Fontan” group compared to the controls, this did not achieve statistical significance. This may be attributed to the duration between CPET and the administration of the questionnaires (~2 years), which likely does not truly reflect their current levels of physical activity. Furthermore, while the IPAQ shows moderate validity compared to accelerometers , in the Indeed, Powell et al. reported that 77% of patients with the “Super-Fontan” phenotype participated in regular physical activity compared to 10% in those with impaired exercise performance . This is2  will hopefully provide more conclusive evidence to aid the development of future exercise training recommendations for people living with a Fontan circulation.It is important to note that the retrospective design of this study can only show association and not causation. A key limitation in this study is the reliability of long-term physical activity questionnaires, which is subject to recall error. However, the reliability of long-term physical activity recall appears to be reasonable, with a previous study reporting an intraclass correlation coefficient of ~0.4 .CPET is a specialized assessment that requires a high degree of clinical expertise to perform and interpret. This may restrict the results of this study to patients in the care of expert centers, which are predominately located in major cities. Indeed, we previously reported that <8% of people in the Australian and New Zealand Fontan Registry had recorded serial CPET documented . TherefoOur study is also limited in sample size, which increases the risk of a type II error, and we may not be able to detect some important associations.2 and/or work rate , and higher overall levels of sport and physical activity participation during childhood and early adulthood. The “Super-Fontan” phenotype exercise response was accompanied by a higher HRR, oxygen pulse, peak circulatory power, and a later anaerobic threshold onset.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by the Royal Children's Hospital Melbourne Human Research Ethics Committee. The patients/participants provided their written informed consent to participate in this study.DT, Yd'U, DC, and RC contributed to the conception and design of the study. DT drafted the manuscript and acquired and analyzed the data. All authors critically reviewed the manuscript, contributed to the interpretation of the results, and approved the submission of the manuscript.DT was supported by a Grant from Additional Ventures, the National Heart Foundation of Australia Vanguard Grant (102277), the Medical Research Future Fund–Cardiovascular Health Mission–Congenital Heart Disease Grant (ARGCHDG000016), and the Paulette Isabel Jones PhD Completion Scholarship.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Alzheimer’s disease (AD) is a degenerative brain disease that is the most common cause of dementia. The incidence of AD is rapidly rising because of the aging of the world population. Because AD is presently incurable, early diagnosis is very important. The disease is characterized by pathological changes such as deposition of senile plaques and decreased concentration of the amyloid-beta 42 (Aβ42) peptide in the cerebrospinal fluid (CSF). The concentration of Aβ42 in the CSF is a well-studied AD biomarker. The specific peptide probe was screened through four rounds of biopanning, which included the phage display process. The screened peptide showed strong binding affinity in the micromolar range, and the enzyme-linked peptide assay was optimized using the peptide we developed. This diagnostic method showed specificity toward Aβ42 in the presence of other proteins. The peptide-binding site was also estimated using molecular docking analysis. Finally, the diagnostic method we developed could significantly distinguish patients who were classified based on amyloid PET images. Alzheimer’s disease (AD) is a neurodegenerative disease that causes the most common form of dementia. It leads to memory loss, cognitive and physical impairment, and finally death ,2. The iThere are many well-researched biomarkers of common neurodegenerative diseases such as amyloid protein, tau protein, and α-synuclein . The amyAβ42 has been detected in numerous experiments, and most of them have used antibodies to detect Aβ42 directly ,22,23. ATo overcome the limitations of using an antibody as a diagnostic probe, a small peptide probe was investigated. Peptide probes have been suggested as alternatives to antibodies in many studies ,30,31,3210 random sequences, and these libraries are sorted through high-throughput screening to identify the most specific phage for the target. The “stickier” phages—those that bind more strongly to the target—occupy the phage pools via affinity selection, while the non-binding phages are removed through consecutive biopanning rounds. Phage display is a useful technique for researching protein–protein interactions, drug discovery, and estimating the epitope of the antigen. It is especially helpful for finding new ligands, such as enzyme inhibitors, receptor agonists, antagonists, and probes that have a targeting moiety [Phage display is the most frequently used biopanning method for screening peptide probes. M13 bacteriophages displaying different peptide sequences on their capsids, according to their genotypes, were used . Phage lg moiety ,39.In this study, we mimicked an ELISA system for Aβ42 detection using a small peptide probe to avoid the disadvantages associated with using an antibody as a diagnostic probe in a sandwich ELISA. We screened novel peptide probes using phage display. The concentrations of Aβ42 in the CSF were estimated and cross-checked with the brain amyloid status based on positron emission tomography (PET) imaging.Aβ42 (ab120301), Aβ40 (ab120479), and anti-β amyloid antibody (ab2539) were purchased from Abcam . 1,1,1,3,3,3-Hexafluoro-2-propanol was purchased from Sigma-Aldrich . Protein G–coated plates were purchased from Thermo Fisher Scientific . Anti-β-amyloid 1–42 (AB5078P) and anti-β-amyloid 1–40 (AB5074P) antibodies were purchased from Merck . M13 phages were purchased from New England Biolabs . The studied Aβ42-binding peptide probe (ABPP) sequences were synthesized by AnyGen . Streptavidin-HRP was obtained from BD Difco Laboratories . 3,3′,5,5′-tetramethylbenzidine (TMB) solution was purchased from R&D Systems . All chemicals were obtained from commercial sources and were of the highest quality available.We added 100 μL of an anti-β-amyloid 1–42 solution  and fluorescein isothiocyanate (FITC)-conjugated antibody (2 μg/mL) in phosphate-buffered saline  to the protein G–coated opaque plates and incubated these for 2 h. Wells were washed three times with PBST (PBS with 0.05% Tween 20) after discarding the unbound reactants. The fluorescence intensity  was estimated after three washes. Then, 100 μL of anti-β-amyloid 1–42 (8 μg/mL) in PBS was added to each well and incubated for 2 h. After antibody fixation, an excessive concentration of Aβ42 (200 ng/mL) was added to each antibody-coated plate for 2 h. Monomerized amyloid solutions were prepared as previously described . The opt10 random sequences was used for the first round of screening. Each round of biopanning was performed under different conditions . Each analyzed phage was amplified by infection with the ER 2738 strain [After four rounds of biopanning, each screened phage presenting its own peptide sequence was analyzed. Sixty phage plaque forming units resulting from biopanning were randomly selected, and the chosen phages were infected with 8 strain .2SO4 was added to the wells to stop the reaction, and the absorbance was measured at 450 nm (OD450) using a microplate reader. To determine the detection range, serially diluted Aβ42 was added to the antibody-coated plates. After washing, the plate was incubated for 1 h with 100 µL of 100 µM peptide. Streptavidin and TMB treatment steps were performed as described above. Based on the measurements, the limit of detection (LOD) for the diagnostic system was calculated according to the formula from the IUPAC: LOD = 3 × SD/slope. To verify the specificity of our diagnostic system, other amyloid proteins were added to the antibody-coated plates. Then, 100 µL of 100 µM of peptide was added to each protein-treated plate and incubated for 1 h. Streptavidin and TMB treatment steps were performed as described above.The studied Aβ-binding peptide probe (ABPP) sequences (S E P Q N I W Q Y L R N) were synthesized by AnyGen . ABPP was conjugated with biotin as a signal reporter, with a purity greater than 95%, as assessed using high-performance liquid chromatography. The binding affinities of peptides were estimated for each Aβ42- and antibody-coated plate. The coating methods followed the protocol described above, except for the use of PBST instead of TBST. After coating the plate with Aβ42, serially diluted peptide was added to the plate for 1 h. After peptide incubation, all wells were washed four times with PBST. Next, the wells were incubated with 100 µL of streptavidin-HRP (1:4000 in PBST) for 1 h. After incubation, the solution in the wells was washed four times with PBST. TMB solution was added to each well for 15 min. Next, 100 µL of 1 M HAnti-Aβ42 and anti-Aβ40 antibodies were applied to the protein G-coated plates and incubated for 2 h. After washing, Aβ42 and Aβ40 were used to consecutively treat each antibody-coated plate for 2 h. Next, 100 µL of 100 µM peptide was added to each protein-treated plate, which was then incubated for 1 h. Streptavidin and TMB treatment steps and absorbance measurements were performed as described above. In addition, two kinds of antibodies—which recognize different epitopes —were added to the protein G-coated plates and incubated for 2 h. The coated plates were washed thrice with PBST. After antibody coating, the same concentration of Aβ40 was administered to the plates, followed by incubation for 2 h. The plates were cleaned three times with PBST. Then, 100 µL of 100 µM peptide was added to each protein-treated plate and incubated for 1 h. Streptavidin and TMB treatment steps were performed as described above.Molecular docking analysis of the peptide against Aβ42 was performed using AutoDock Vina . The binPBS and human serum were used as solutions for the spiking test. Each of the Aβ42 and Aβ40 stock solutions was diluted using PBS, and the mixture was diluted using human serum. The diluted samples were added to antibody-coated plates and incubated for 2 h. The binding conditions used for the antibody coating and the peptide, streptavidin, and TMB treatment steps are described above. To calculate the recovery ratio, we divided the signals of the mixtures by the signals of Aβ42.18F)-florbetaben amyloid PET, and CSF analysis. The participants who were included in the study met the following criteria: they (1) were aged over 40 and under 90 years; (2) showed cognitive impairment, which included subjective cognitive decline (SCD), mild cognitive impairment (MCI), and dementia, and was defined by subject memory impairment reported by the patients or caregivers, activities of daily living, as judged by a physician, and objective memory decline below the 16th percentile (−1 standard deviation) for the demographically matched norms determined via neuropsychological testing; and (3) showed no evidence of structural lesion on a brain MRI that could influence cognitive function. All PET images were obtained using Discovery 690, 710, and 690 Elite PET/computed tomography scanners . Amyloid PET images were acquired for 20 min, beginning 90 min after injection of 300 ± 30 MBq of 18F-florbetaben. Two neurologists (H.J.K. and J.H.L) and two nuclear medicine physicians reviewed the PET scans according to the predefined regional cortical tracer binding and brain amyloid plaque load (BAPL) scoring system [ApoE genotype was identified after extracting genomic DNA from the venous blood.This study consecutively and prospectively enrolled 98 participants who visited the memory clinic of Asan Medical Center, Seoul, South Korea, from June 2018 to July 2020. All participants or their proxies provided informed consent. All participants underwent brain MRI, detailed neuropsychological testing, fluorine-18  as a formal test to accurately describe their cognitive status as SCD, MCI, or dementia . The SNSThe concentration of Aβ42 in the CSF was examined using a commercial ELISA kit  according to the manufacturer’s instructions. Briefly, the CSF samples were added to human Aβ42 monoclonal antibody-coated microplates and incubated for 2 h at 2–8 °C. The unbound substances were washed away, and the molecules presenting only Aβ42 were attached to the capture antibody. Then, the captured Aβ42 was detected using enzyme-linked monoclonal antibodies for another 2 h at 2–8 °C. Substrates were added to the microplates, and color development was measured using a microplate reader at a wavelength of 450 nm.CSF samples from patients were added to antibody-coated plates and incubated for 2 h. The binding conditions used for antibody coating and the peptide, streptavidin, and TMB treatment steps are described above.t-test was used to analyze the age, educational level, disease duration, MMSE, CDR, neutrophil, monocyte, ESR, C-reactive proteins (CRP), and low-density lipoprotein (LDL) levels. The χ2 test was used to analyze the sex distribution, apolipoprotein E (ApoE) ε4 allele, diabetes mellitus (DM), hypertension (HTN), and hyperlipidemia. For the ELISA and ABPP sensitivity and specificity tests, Student’s t-tests and ROC analyses were used.GraphPad Prism 6 was used for statistical analysis. Statistical analyses of the demographics of the participants were conducted as follows. Student’s As the concentration of anti-Aβ42 antibody increased, the fluorescence intensity of the FITC-labeled antibody decreased. Due to occupation by a non-labeled anti-Aβ42 antibody, FITC-labeled antibody could not bind to the plate. Saturation of the anti-Aβ42 antibody was observed at 8 μg/mL B.D) for the ABPP was 17.47 ± 2.98 μM  than in the Aβ− group (23.1%). The frequency of DM was higher in the Aβ− group than in the Aβ+ group. Serum ESR was higher in the Aβ− group, and LDL levels were higher in the Aβ+ group. Other demographic factors, including sex distribution, disease duration, educational level, frequency of HTN, and hyperlipidemia, were not significantly different between the two groups.The detailed demographic and clinical characteristics of the participants are presented in Amyloid PET imaging is one of the main medical approaches used to diagnose AD. The participants were divided into two groups—amyloid-positive and amyloid-negative—based on the existence of deposited amyloid proteins . ELISA aIn this study, we developed an Aβ42 detection system using a peptide as a signaling probe. Although sandwich ELISA is an easily accessible method for measuring target concentrations, it is not very precise when detecting Aβs. To overcome the limitations of the general sandwich ELISA, which could not be used to analyze the lower molecular weight amyloid aggregates, we attempted to develop a peptide probe for Aβ42. Unlike sandwich ELISA, our developed method uses antibodies only once to capture the target.The peptide was screened using phage display as a signaling probe. For biopanning, Aβ42 was fixed onto the plate and captured by the antibody. Then, phage binding, washing, elution, and amplification for the next screening rounds proceeded under harsh conditions. Due to the use of an antibody as a capture probe, our system shows specificity toward Aβ42. For the fixation of antibodies on the plate surface, we used protein G-coated plates, which increased the efficiency of antibody fixation by helping the antibodies face the same direction . FluoresAmong the screened peptides, we selected one sequence based on its solubility and synthesized it. Another sequence had too few hydrophilic residues, which affected its solubility. Because hydrophobic sequences require organic solvents, we synthesized the most hydrophilic sequence . The synR2 value indicates that our standard curve showed a good linear relationship between the OD450 value and Aβ42 concentration [To quantify Aβ42, we performed regression analysis. The high ntration . For accntration . To veri18F-florbetaben detected cortical amyloid aggregates in patients. All participants were divided into two groups based on their amyloid PET results: amyloid-positive and -negative. Our dataset included 24 subjects aged <65 years who had an early onset of symptoms. Therefore, the apparent difference in age and baseline cognitive function between the two groups can be attributed to these early onset patients. The higher number of ApoE ε4 allele carriers in the amyloid-positive group was also consistent with that in previous studies [Fluorine-18–florbetaben amyloid PET is a useful clinical tool for the visualization of amyloid plaques in patients with AD . The PET studies . Further studies . As desc studies . This meIn this study, we identified the peptide sequence that bound most strongly to the monomeric state of Aβ42, and our screened peptide showed specific binding to only Aβ42 using the developed system. The binding sites were also estimated and matched using molecular docking analyses. Finally, patients with amyloid depositions in the brain were distinguished using our peptide-based diagnostic method. Our diagnostic method still has problems, such as its sensitivity and specificity; however, our peptide and the diagnostic method are helpful for several clinical research areas in the AD field because of their potential to detect the monomeric state of Aβ42. Using a peptide has several benefits—such as its small size, non-immunogenicity, stability, and ease of modification—not only in terms of research but also in commercial applications. These advantages of using a peptide probe as an alternative to antibodies appear to be relevant to AD research.


To investigate the MRI features and clinical significance of hepatic epithelioid hemangioendothelioma (HEHE).Clinical records and MRI findings were retrospectively evaluated in nine HEHE patients from May 2010 to January 2020.p < 0.001). All lesions exhibited a two-layer appearance on diffusion-weighted imaging (DWI), with hyperintensity at the periphery and a slightly high signal at the center (except for those with a single layer on T2WI). The “vascular penetration sign” was observed in most lesions, and the blood vessels of 112 lesions (92.56%) were portal vein branches, and five (4.13%) were hepatic vein branches. Pulmonary metastasis was found in three patients with the “vascular penetration sign” of hepatic vein branches.There were 121 lesions in nine patients with a predominantly peripheral distribution. Five lesions (4.13%) in two patients (22.22%) had evidence of capsular retraction, and three patients had lung metastasis (33.33%). Dynamic contrast-enhanced MRI showed progressive enhancement, mainly in two ways: ring enhancement with hypovascularity in four patients (44.44%) and ring enhancement with hypervascularity in five patients (55.56%). Imaging demonstrated a multilayer ring appearance, which was typically observed on T2-weighted imaging (T2WI). The most common appearance consisted of two layers of varying signal, with some images displaying up to four layers. There were significant differences in the size of lesions between different layers of multilayer ring appearance (The multilayer ring appearance on T2WI, the “vascular penetration sign”, and the two enhancement patterns may be of great significance in the diagnosis and treatment of HEHE. The “vascular penetration sign” of hepatic vein branches may indicate extrahepatic metastasis. Hepatic epithelioid hemangioendothelioma (HEHE) is a rare vascular malignancy of the liver that carries a low malignant potential (incidence rate of 1/1 million population) . OriginaAfter early diagnosis, effective treatment options include surgical resection and liver transplantation. However, most patients are diagnosed with HEHE in the final stage when surgical resection is not possible , 11, 12.Imaging examination is one of the main methods for diagnosing liver lesions , 17. HowNumerous studies have explored the imaging features of HEHE and demonstrated several characteristics, such as early peripheral ring enhancement, capsule retraction, and the “lollipop” sign –23. HoweClinical records and MRI findings of all initially diagnosed HEHE patients admitted to the Fifth Medical Center of PLA General Hospital from May 2010 to January 2020 were reviewed. Nine patients Table 1 2 with the following parameters: TR/TE, 4615/60 ms; matrix, 160 × 128; 6–10-mm thickness with a layer spacing of 1–2 mm; FOV, 34 × 40–40 × 40 cm. Three-dimensional fat-saturated T1-weighted dynamic contrast enhancement sequences were also performed . The injection rate of the high-pressure injector was 1.5–2.5 ml/s at a dose of 0.1 mmol/kg. The parameters were as follows: TR/TE, 3.9/1.7 ms; matrix, 256 × 256–320 × 256; 5-mm thickness with an interpolated section thickness of 2.5 mm; FOV, 34 × 40–40 × 40 cm.MRI examinations were performed on a 3.0-T system  HDXt). For T2WI, respiratory-triggered fat-suppressed fast spin-echo (FSE) was used with the following parameters: repetition time (TR)/echo time (TE), 9,000/81.9 ms; matrix, 256 × 256–320 × 256; 6–10-mm thickness with a layer spacing of 1–2 mm; field of view (FOV), 34 × 40–40 × 40 cm. Then, transverse T1-weighted in-phase and out-of-phase sequences were performed using the following parameters: TR/TE, 3.8/ ms; matrix, 256 × 256–320 × 256; 5-mm thickness with an interpolated section thickness of 2.5 mm; FOV, 34 × 40–40 × 40 cm. Transverse breath-hold diffusion-weighted imaging (DWI) was obtained using b-values of 0 and 800 s/mmThe LightSpeed volume CT (VCT) spiral CT scanner by GE (United States) was used. Patients were scanned in a supine position with the head advanced and the breath held at the end of inspiration. CT scanning parameters were as follows: tube voltage, 100–120 kV; tube current, 150 mA; pitch, 0.98 mm; matrix, 512 × 512; slice thickness, 5.0 mm; FOV, 350 × 350 mm; multiplanar reconstruction with a thickness of 1.25 mm. For the lung standard window, the window width was 1,500 HU, and the window level was −500 HU. For the mediastinal window, the window width was 350 HU, and the window level was 50 HU.All images were independently reviewed by two radiologists who had 10 and 8 years of experience in the interpretation of abdominal MR images. Final decisions were reached by consensus. The “vascular penetration sign” was defined as the penetration of the terminal branch of the portal or hepatic vein from the edge to the interior of the tumor that extends a certain distance on dynamic contrast enhancement.All patients underwent liver biopsy, which included six percutaneous ultrasound-guided biopsies and three CT-guided biopsies. The histopathological evaluation included routine H&E and immunohistochemical staining. Endothelial markers included CD31, CD34, and a factor VIII-related antigen. Pathological examinations and specimen handling were performed by experienced liver pathologists.p < 0.05 was considered statistically significant.SPSS 13.0 software was used for data analyses. Continuous variables are expressed as means ± SDs. Comparisons between groups were performed using one-way ANOVAs. A There were a total of nine patients Table 1,All nine patients underwent abdominal MRI and lung CT. Eight patients had multiple lesions , and one patient had a single lesion . There were a total of 121 nodules with a predominantly peripheral distribution Table 2.All patients showed a multilayer circular structure on MRI, which we called a “multilayer ring appearance”. This feature was seen most clearly in T2WI Figure 1A multilayer ring appearance consisting of a core was more commonly seen in the two-layer structure but could be observed in up to four layers. The layer structure of nodules varied across patients. For example, the MRI of patient 8 (32 lesions) showed several lesions with a two-layer ring appearance (18 lesions) and other lesions with three layers (14 lesions). The multilayered ring appearance showed an alternation between high and low signal intensity , the DWI showed a target appearance that consisted of a core with a slightly hyperintense signal and a peripheral halo with high signal intensity  were portal vein branches or hepatic veins. In patient 2, among the five lesions, the blood vessels of two lesions were portal vein branches, and those of three lesions were hepatic vein branches. In patient 3, among the four lesions, the blood vessels of three lesions were portal vein branches, and those of one lesion included both hepatic vein and portal vein branches. In patient 4, among the five lesions, the blood vessels of four lesions were portal vein branches, and those of one lesion were hepatic vein branches. The chest CTs of these three patients indicated lung metastasis. All of the “vascular penetration signs” in the other six patients involved portal vein branches rather than hepatic vein branches and were not lung metastases. Thus, the “vascular penetration sign” with hepatic vein branches may be related to lung metastasis.Dynamic contrast-enhanced MRI Figure 4HEHE is a rare vascular low-grade malignant tumor of the liver. It is frequently clinically misdiagnosed. We retrospectively studied nine patients with pathologically proven HEHE. Patients in this study were mainly male, which was different from previous reports , 9. The Several studies have suggested that nodule coalescence and capsular retraction are characteristics of HEHE , 20, 24.Dynamic contrast-enhanced images showed that progressive enhancement was exhibited by all patients and followed two major patterns. Four patients  showed ring enhancement with hypovascularity, and five patients  showed ring enhancement with hypervascularity. This feature has been reported in previous studies .The “multilayer ring appearance” is considered a characteristic of HEHE. Previous studies have termed this ring structure as a “target-like sign” , 21, sucp < 0.001). This suggested that the number of layers in the “multilayer ring appearance” of the lesion is related to the growth of the lesion, where the larger the lesion, the higher the number of layers. Therefore, further pathological studies are needed to better understand the “multilayer ring appearance” of HEHE. However, irrespective of the number of layers of ring structure in the lesion, the DWI images always showed a two-layer target structure with a hypointense inner layer and core and a hyperintense rim.We analyzed the multilayer ring structure of 121 lesions in nine patients and found that most lesions had a two-layer structure with a maximum of four layers. Several patients exhibited two patterns of the “multilayer ring appearance” (mainly differences in the number of layers). Moreover, we compared the appearance of the feature using different imaging sequences and found that the “multilayer ring appearance” could be detected more clearly in T2WI. Notably, there were significant differences in the size of lesions between different layers of the “multilayer ring appearance” (Imaging features are often related to the molecular mechanism of the tumor. The occurrence and progression of liver tumors have complex molecular and cellular changes, which can be useful for understanding the biological behavior and the treatment of tumors –30. TherThe relationship between lesions and blood vessels may be another feature of HEHE. Several studies have suggested that the “lollipop sign” , 21, whiInterestingly, 3/9 patients had extrahepatic metastases, which were mainly found in the lungs. These three patients all had the “vascular penetration sign” of the hepatic vein. Tumor cells may enter the inferior vena cava through the hepatic vein and subsequently reach the lungs, which may cause pulmonary metastasis. Our MRI analysis of the other six patients with no pulmonary metastasis showed that all the blood vessels of lesions with the “vascular penetration sign” were portal veins rather than hepatic veins. Therefore, we speculated that the “vascular penetration sign” of the hepatic vein is related to lung metastasis; moreover, tumor cells may infiltrate the vascular walls to metastasize. An international multicenter study showed positive macrovascular invasion in surgical specimens and extrahepatic disease .In conclusion, this study investigated the MRI findings of nine patients with the rare disease HEHE in combination with clinical practice and found distinctive features, which included the “multilayer ring appearance”, “vascular penetration sign”, and two patterns of enhancement. The “vascular penetration sign” of the hepatic vein was highly indicative of lung metastasis. These findings are of great significance for the diagnosis and treatment of HEHE; moreover, they provide insight into the development and prognosis of the disease. Further comparative studies of pathology during different stages of disease progression are needed to gain a better understanding of HEHE.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by The 5th Medical Center of PLA General Hospital. The patients/participants provided their written informed consent to participate in this study.XG performed the statistical analysis and drafted the manuscript and the study design. XG, YY, WZ, HZ, YZ, KZ, HK, LY, YC, YB, and ZZ performed the MRI of the patient studies. XG, HZ, WZ, YZ, and KZ take responsibility for the data collection and sorting.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study was conducted in a general population of schoolchildren in Sweden, with the aim to assess the psychometric properties of a generic preference-based health related quality of life (HRQoL) instrument, the Swedish Child Health Utility 9D (CHU9D), among schoolchildren aged 7–15 years, and in subgroups aged 7–9, 10–12 and 13–15 years.In total, 486 school aged children, aged 7–15 years, completed a questionnaire including the CHU9D, the Pediatric quality of life inventory 4.0 (PedsQL), KIDSCREEN-10, questions on general health, long-term illness, and sociodemographic characteristics. Psychometric testing was undertaken of feasibility, internal consistency reliability, test–retest reliability, construct validity, factorial validity, concurrent validity, convergent validity and divergent validity.The CHU9D evidenced very few missing values, minimal ceiling, and no floor effects. The instrument achieved satisfactory internal consistency (Cronbach’s Alfa > 0.7) and strong test–retest reliability (r > 0.6). Confirmatory factor analyses supported the proposed one-factor structure of the CHU9D. For child algorithm, RMSEA = 0.05, CFI = 0.95, TLI = 0.94, and SRMR = 0.04. For adult algorithm RMSEA = 0.04, CFI = 0.96, TLI = 0.95, and SRMR = 0.04. The CHU9D utility value correlated moderately or strongly with KIDSCREEN-10 and PedsQL total scores (r > 0.5–0.7). The CHU9D discriminated as anticipated on health and on three of five sociodemographic characteristics .This study provides evidence that the Swedish CHU9D is a feasible, reliable and valid measure of preference-based HRQoL in children. The study furthermore suggests that the CHU9D is appropriate for use among children 7–15 years of age in the general population, as well as among subgroups aged 7– 9, 10–12 and 13–15 years. Economic evaluations, including cost-utility analysis (CUA), have a central role in healthcare decision-making . CUA is Existing Pediatric MAU instruments are mostly adapted from adult instruments or developed from the perspective and preferences of adults –4. Such The Original English version of the CHU9D has demonstrated sound psychometric properties in 7 to 11-year-olds in the UK  and in 1The CHU9D has been translated into Swedish. The current study is the first to investigate the psychometric properties of the Swedish CHU9D.This study aimed to investigate the feasibility, reliability, and construct validity of the CHU9D among school-aged children attending grades 1–9 in Sweden (ages 7–15 years), and in subgroups attending grades 1–3, 4–6 and 7–9 .The study utilized a convenience sample of children attending grades 1–9 (ages 7–15 years) in four elementary schools in a city in Northern Sweden with around 89,000 inhabitants. Around 9000 of these are 7–15 years old. A minimum of 50 children were included from each grade (1–9), resulting in a general population sample of at least 150 children from each of the three elementary school stages: grades 1–3; 4–6; and 7–9 . The one exclusion criteria used was (children) not being fluent in Swedish.Headmasters and class-teachers were informed about the study. Following their consent, children were informed about the study in class and parents received written information. Informed consent to participate was sought from the child. In children below 15 years, also parental consent was requested. After obtained consent, children attending school the day of the survey, filled in a questionnaire in school, assisted by a specially trained research assistant (school nurse). Approximately 59% of the approached students participated in the study. Parents of children in grades 1–3 filled in a short questionnaire at home. In the younger age groups (grades 1–3), the research assistant read the questions and response alternatives aloud, one by one. In each grade, approximately half of the participating schoolchildren filled in the CHU9D again, 7–15 days after the first assessment. Whole classes were randomly selected for the second assessment. Teachers facilitated the process, and were present, but not actively involved in the data collection.The children answered a questionnaire including three measure of HRQoL, the CHU9D, KIDSCREEN-10 , 19 and The preference-based CHU9D measures HRQoL by nine-dimensions: worried, sad, pain, tiredness, annoyed, schoolwork/homework, sleep, daily routines, and ability to join in activities. Recall time is today or last night, and each dimension has five severity levels , 22. RepKIDSCREEN-10 addresses 8–18-year-old children and captures overall non-preference based HRQoL based upon 10 underlying dimensions: feeling fit and well, full of energy, sad, lonely, and having enough time for one-self, being able to do what you want at spare-time, being treated fairly by parent(s), having fun with friends, doing well in school, and being able to pay attention (in school) . KIDSCREThe non-preference based PedsQL has 23 age-specific items that capture 4–18-year-old children’s overall HRQoL, as well as the underlying psychosocial (15 items) and physical (8 items) dimensions . The psySelf-reported general health was assessed by the question “How do you feel in general”. The question has five response alternatives, which were merged into three categories: not good/fair (named not good onwards), good, and very good/excellent (named very good onwards). This question was only assessed in grades 2–9.Long-term illness/disability was studied by asking about the presence of seven specified pediatric chronic or long-term health problems  and one alternative named “others”. In grades 1–3, this information was obtained from the parents and in grades 4–9 from the child. Children with at least one chronic or long-term health problem were classified as having a long-term illness/disability.Sex, grade, children’s and parent’s country of birth, and custody arrangement were measured by specific questions. In grades 1–3, parents provided information about country of birth.Socio-economic status was measured by the Family Affluence Scale (FAS) . FAS assTo assure equivalence of CHU9D scores at the two assessments used for the test–retest analyses , children were asked “Has anything out of the ordinary happened that makes you feel better or worse today”. The response alternatives were no vs yes, please describe. The answers were independently reviewed by two of the authors, (KL and MV) to determine if any child needed to be excluded out of the test–retest analysis due to having a major life event either at the first or second time of participation. If there were any uncertainties, a third author (SP) was consulted.Differences in CHU9D utility scores were estimated using multivariate tests of means for the one-sample test when applying the child and the adult algorithms, respectively.Feasibility was examined by estimating floor and ceiling effects and the proportion of missing values. The Cronbach’s coefficient α was used to evaluate internal consistency reliability, with values ≥ 0.7 considered acceptable for group and ≥ 0.9 for individual comparisons [test–retest reliability) using Intraclass-, Canonical- and Spearman’s correlations for scale comparisons, along with Spearman correlations and Weighted Kappa statistics for dimension comparisons. Correlations of 0.00–0.19 were considered very weak, 0.20–0.39 weak, 0.4–0.59 moderate, 0.60–0.79 strong and 0.80–1.00 very strong [parisons . Furthery strong . Kappa vy strong .Construct validity was assessed through confirmatory factor analysis testing the hypothesized one-factor structure of the CHU9D . The following model fit indices and cut-offs were used to confirm model adequacy: comparative fit index (CFI) and Tucker-Lewis index (TLI) values ≥ 0.90 (acceptable fit) or ≥ 0.95 (excellent fit) [Construct validity was also explored by comparing the total CHU9D utility scores to the total KIDSCREEN-10 and the PedsQL scores, using Spearman’s correlations . Spearman’s correlations were furthermore used to test whether the conceptually alike dimensions of the CHU9D and the other HRQoL instruments  and difference between correlations of conceptually alike and dislike dimensions were explored .ent fit) , root meent fit) , and staent fit) . Construconstruct validity was assessed by the known-groups method, i.e. by comparing CHU9D utility scores depending on sex, school stages , parental country of birth (Sweden: none-one-both parents) custody arrangement (living with both parents-or not), family affluence , general health status (not good-good-very good), having a long-term illness or disability (yes–no). Differences between groups were estimated using Mann–Whitney U-test and Kruskal–Wallis test. Lower utility scores were anticipated in girls  and older age groups, and in those with a foreign background, not living with both parents, having lower family affluence, not holding a good general health, or having a long-term illness or disability [Finally, sability , 31–33.p < 0.05).The analyses were performed for grades 1–9 in total, and separately for each of the three school stages studied, using Stata version 16.1 . Relationships mentioned in the result section are significant at the 95% level . Measured by KIDSCREEN-10 and PedsQL, the corresponding numbers were 41.17 (SD ± 6.10) and 82.12 (SD ± 12.94), respectively.Data was collected from 486 children. Of these, 473 (97%) answered all CHU9D questions and thus, were included in the analysis. These participants were evenly distributed between the three studied school stages, but included slightly less girls than boys Table . The greNo child reported the worst possible health state for all nine dimensions (no floor effect), while 8.5% reported the best possible health state, i.e. no problems, for all dimensions (ceiling effect) Table . StratifStudying each of the nine dimensions separately, floor effects were generally below 2%, and with a few exceptions, this was true across all three school stages. The “tiredness” dimension, however, revealed an overall floor effect of 9%, varying from 5% in grades 4–6 to 16% in grades 7–9. Ceiling effects, on the other hand, overall ranged from 21% (tiredness) to 77% (sadness) and the general pattern of lower ceiling effects by higher school stage was seen for each of the nine dimensions.In total, 255 children filled in the CHU9D twice. Of these, 13 children were excluded from the test–retest analyses, 11 because of a major event happening between rating occasions and 2 children had missing values in several dimensions at the second assessment. Thus, 242 children were included in the test–retest analyses, 73 from grades 1–3, 81 from grades 4–6 and 88 from grades 7–9.At scale level, Canonical, Spearman’s and Interclass correlations all showed strong (> 0.7) or very strong (> 0.8) correlations between the two occasions Table . SimilarWhen comparing the individual dimensions-scores from the test–retest occasions, one by one, correlations were moderate or strong (0.41–0.71), while kappa-agreement were moderate or close to moderate for 6 dimensions 0.39–0.54) and fair for 2 dimensions (“pain” 0.32 and “annoyed” 0.35) . Furthermore, as seen in Table r > 0.6 and 0.7, respectively). In grades 1–3, these correlations were moderate with r just above 0.5 (both KIDSCREEN and PedsQL).Strong correlations were found between the total score of CHU9D  and the total scores of KIDSCREEN (0.61/0.62) and PedsQL, (0.62/0.61). Stratified analyses showed that, in the two older school stages, the CHU9D total scores correlated strongly with the KIDSCREN-10 and PedsQL scores (r > 0.4), or close to moderate, relationships to the conceptually alike KIDSCREEN-10 and PedsQL dimensions  than to the physical functioning dimension (r 0.31). Another exception was that the dimension on abilities to join in activities showed the strongest relationship with the KIDSCREEN-10 dimension “fit and well” (r 0.33). and not the expected dimension regarding ability to do desirable spare time activities (“able to do things” r 0.26). In each of the three separate school stages, the results overall follow the same pattern as describes above, although correlations seem to be slightly weaker among the youngest children.The individual nine CHU9D dimensions generally demonstrated moderate , and separately for children in grades 1–3, 4–6 and 7–9 .Very few missing values, minimal ceiling, and no floor effects support the general feasibility of the Swedish CHU9D. However, ceiling effects were relatively high for several of the underlying CHU9D dimensions. This is not surprising given that many children are expected to be at good health in general populations. Similar results have been shown when using the English, Danish and Chinese CHU9D in general populations of school-aged children , 33, 36.The reliability of the Swedish CHU9D was supported by strong test–retest correlations and agreements, along with established internal consistency. Again, this is in line with the result of studies using the English, Danish and Chinese CHU9D in their cultural contexts , 36, 37.The current study confirms the proposed one-factor structure of the CHU9D. In the absence of a gold standard for HRQoL measurement, it is not possible to prove conclusively that this factor measures HRQoL. However, we found a strong correlation between the total scale score of the CHU9D and two HRQoL instruments with demonstrated reliability and validity (KIDSCREEN-10 and PedsQL). Furthermore, the strongest correlations were seen between the CHU9D dimensions and the conceptually overlapping KIDSCREEN-10 and PedsQL dimensions, while correlations were weaker for non-overlapping concepts. Other studies have shown similar results when comparing the original English CHU9D to KIDSCREEN-10 and PedsQL , 38 or tAlthough we and other researchers , 33, 38 Consistent with studies from other countries, , 33, 36 The CHU9D was initially developed for ages 7–11 years . Our stusadness, worries, being annoyed) and impairment in daily functioning  but comparably higher weight on health states dominated by physical impairment  [Our results confirm earlier findings showing that CHU9D utility scores are higher when applying the adult as compared to the child algorithm , 39. Thiroblems) . Thus, tnot good” and a “very good” general health, we found utility scores difference to be twice as high when applying the child algorithm as compared with the adult algorithm (mean-score difference: 0.33 vs. 0.17). Such differences suggest that the choice of algorithm may have the capacity to influence interpretations of future economic evaluations of pediatric health interventions, highlighting the importance of this choice. We acknowledge some limitations. Although including only fluently Swedish speaking children diminishes biases due to language barriers, which is a strength, it may also have biased the discriminative analysis regarding SES and ethnic origin. Also, given that the study was based on a convenience sample, it cannot provide normative information about HRQoL levels. Another limitation is that this general population study with self- and parent-reported health, could not evaluate the applicability of the CHU9D in clinical population. Likewise, the study design did not allow evaluations of responsiveness to change in health status over time. In addition, the long-term illness/disability measure was based on self-report by children or parents, and were not confirmed via medical records.Notably, we found that the algorithm used influenced the size of between-groups differences. Comparing for instance those with a “This study provides support that the Swedish CHU9D is a feasible, reliable and valid measure of HRQoL that holds psychometric qualities comparable to those of the original English CHU9D. The study furthermore, suggests that the CHU9D is appropriate for use among 7–15-year-old children in the general population, as well as among subgroups aged 7– 9, 10–12 and 13–15 years. To provide further support for the CHU9D as a useful health outcome measure in health economic evaluations, future studies should investigate the performance of the CHU9D in clinical samples. Longitudinal studies are also needed to test the instruments sensitivity to change.


Solid-state electrolytes are a promising family of materials for the next generation of high-energy rechargeable lithium batteries. Polymer electrolytes (PEs) have been widely investigated due to their main advantages, which include easy processability, high safety, good mechanical flexibility, and low weight. This review presents recent scientific advances in the design of versatile polymer-based electrolytes and composite electrolytes, underlining the current limitations and remaining challenges while highlighting their technical accomplishments. The recent advances in PEs as a promising application in structural batteries are also emphasized. Global development is highly dependent on energy. Considering the impact of fossil fuels, global warming, and widespread pollution, the need for green, renewable, and alternative energy sources and storage systems is vital . The celAt the end of the 20th century, and driven by the emerging market for wireless technologies, the demand for efficient and safe rechargeable batteries increased and lithium-based batteries became the go-to solution to fulfil most requirements . LithiumDepending on their physical state, electrolytes can be classified as liquid or solid. Solid electrolytes are typically classified in two subtypes: (i) organic (polymer-based) and (ii) inorganic. On the other hand, polymer electrolytes can be subdivided into solid-polymer electrolytes (SPEs) and gel-polymer electrolytes (GPEs). In the former, the polymeric matrix is a solid ionic conductor itself, and the latter comprises the incorporation of a common liquid electrolyte into a polymer, aiming at improving its properties. Besides, when the incorporation of fillers is accomplished, a new class of electrolytes arises that is titled “composite-polymer electrolytes” (CPEs).Aside from the obvious morphological differences, the expected performance of each type of electrolyte is also noticeably distinct. −3 to 10−2 S·cm−1 at room temperature (RT). Nevertheless, hazards (such as high flammability) and poor recyclability still restrict their use in a wide range of technological applications +), imidazolium ([EMIM]+), piperidinium ([BMPip]+), quaternary ammonium ([NR4]+) with anions as hexafluorophosphate ([PF6]−), tetrafluoroborate ([BF4]−), dicyanimide ([N(CN)2]−), and bis[(trifluoro methyl)sulfonyl]imide ([CF3CONCF3SO2]−), among others.Plasticizers, with a low molecular weight, organic solvents, or ionic liquids, can increase the amorphous content of a polymer and, thus, increase the ionic conductivity . For exao et al.  developen et al.  by prepa+ transport is typically correlated to the motion of the polymer chain segments and mainly occurs in the amorphous phase of the SPE. Some strategies have been studied and reported in the literature to decrease crystallinity. As mentioned before, both plasticizers and inorganic fillers can increase the amorphous content. On the other hand, the reduction of Li+ diffusion pathways allied with the enhancement of the cell capacity makes the idea of the incorporation of the inorganic filler very attractive . T. T86]. Tuctivity b, which −4 S·cm−1 and a wide electrochemical window up to 5.4 V (against Li/Li+). This optimized membrane (XP-20) contained a mixture of poly(ethylene glycol) methyl ether methacrylate (PEGMEA), LiTFSI, and poly(ethylene glycol) dimethyl ether (PEGDME) at a 5:3:4 weight ratio and a 20 wt.% ratio of the XVIm-TFSI crosslinker without the addition of plasticizers. As shown, the effect of the imidazolium crosslinker proved to enhance not only the ionic conductivity of the membranes but also the compressive elastic modulus, as well as its thermal stability, compared to the PIL-based copolymer, without crosslinking sites (P-20).As mentioned earlier in this review, another approach applied to SPE development consists of using crosslinked structures by providing enhancement of mechanical properties through permanent covalent bonds formed between two types of monomers. Moreover, crosslinking is known to reduce the crystalline domains of a polymer, resulting in enhanced conductivity, as the amorphous regions enable higher ionic conductivity . The pos4 cell naturally showed a good specific capacity of 160 mAh·g−1 at a 0.2 C rate while maintaining 93.8% capacity retention after 150 cycles, a characteristic that was not present in the copolymer without the imidazolium crosslinker, where the performance of the cell continuously faded over cycling.In addition, crosslinking successfully improved the membrane cycling stability against dendrite formation due to reduced polarization, stable SEI formation, and superior mechanical strength. The Li/LiFePO2-PEG-NH2) and benzene-1,3,5-tricarbaldehyde (BTA) dynamic imine bonds (−3 S·cm−1 at 30 °C (−1 (5 C rate) and 97.8% capacity retention after 125 cycles  solution mixed with LiTFSI salt, the resulting GPE containing an optimal 30 wt.% PC + 20 wt.% LiTFSI delivered an ionic conductivity of 3.5 × 10sticizer c. It is Li+. In these materials, only 20% of the ionic conductivity is due to the Li mobility, resulting in an enhancement of the ionic concentration gradient and, consequently, promoting an increase in cell polarization during the discharge process. This effect limits the amount of current available and decreases the lifetime of the battery [To develop new types of polymeric electrolytes exhibiting high conductivity and good mechanical properties, dendrite suppression is a fundamental aspect to consider during the material design to ensure good safety of the overall system. According to the current state of the art, SPEs show dual ionic conduction, as lithium ions and their respective counter ions migrate during charge and discharge cycles between the cathode and anode, displaying lower t battery .+ ions. The use of single-ion polymer electrolytes (SIC), which have a tLi+ close to the unity, has proved to be an efficient method for suppressing dendrites and increasing the battery’s life and safety, as the anion immobilization allows a free migration of Li+ ions and consequent decrease in cell polarization. Owing to the potential of SIC, a variety of reviews reported state-of-the-art developments over the last years focusing on this thematic [This main drawback in traditional SPEs can be overcome by blocking the migration of Li counter ions, typically carried out by tethering the counter ions’ moieties to the polymer backbone, promoting the free migration of Lithematic ,78,113. −6 S·cm−1 and a high tLi+ of 0.83, characteristic of a SIC polymer. The low RT conductivity of the copolymer was mostly due to the high association of Li ions to the tethered anion moieties, which resulted in poor ion solvation. To overcome this challenge, the authors proposed the addition of a plasticizer.Several polymers containing sulfonate have been developed due to their easy preparation and acceptable delocalized structure, and thus provide reasonable electrochemical properties and improve safety. Yubin et al.  reported−5 S·cm−1; good mechanical performance, namely, tensile strength (>130 kPa) and stretching (>4000%); and enhanced flame retardancy. The results also evidence that a decrease in the gT occurred with the addition of ionic liquid owing to the lower crystalline regions, resulting in more Li+ migration and increasing the conductivity.In recent studies, self-healing and stretchable polymer electrolytes exhibiting increased flame retardancy properties were developed by free radical polymerization of 2,2,3,4,4,4-hexafluorobutyl methacrylate (HFBM) and sulfobetaine (SBMA) monomers with sulfonate-containing groups . Differe4 cell displayed a high discharge capacity of 144.8 mAh·g−1 (0.2 C rate), with 82% stability over 100 cycles. The self-healing ability was attributed to the imidazole moieties present in the EMI-TFSI ionic liquid, which formed ion–dipole interactions with the fluorine atoms of the polymer side chain, resulting in an impressive total repair and full recovery of its electrochemical and mechanical properties in less than 60 min.The assembled Li/LiFePOIn addition, Li et al.  were abl−3 S·cm−1 ionic conductivity at RT, as well as a high tLi+ of 0.64 and good anodic compatibility.Subsequently, the desired GPE membrane was prepared by electrospinning, followed by swelling in a 1 M LiTFSI-based liquid electrolyte. The obtained findings show that the Li-SPOD membrane presented a high degree of porosity, thus being capable of a higher liquid uptake compared to the traditional polypropylene (PP) separator . The synthesis of a highly delocalized single-ion conductive structure was confirmed by electrochemical measurements, where the Li-SPOD GPE exhibited a superior 2.03 × 104 cell delivered an initial discharge capacity of 125 mAh·g−1 (2 C rate) and was capable of significantly suppressing the formation of dendrites on the metallic Li anode after 300 cycles, in comparison with the cell assembled with a PP separator that did not deliver the same suppression capacity, as can be seen in Moreover, the freestanding electrospun membrane displayed a decent tensile strength of 14.2 MPa and good thermal stability up to 472 °C. The Li/LiFePOLi+, has been achieved, making this type of material promising for future applications. Bis(benzene sulfonyl)imide was selected in several studies for the development of different SPEs, promoted by the higher delocalization of the bis(sulfonylimide) bond covalently linked to a benzene ring at both ends, with the effect of this polyanion on the conductivity and tLi+ of the SPE being notorious. Recently, in 2019, Cao et al. [−4 S·cm−1 at RT and high tLi+ of 0.97, without the incorporation of plasticizers imide-based SPEs, as well as dendrite suppression capacity due to the superior t2)2 groups conferred superior mechanical performance to the membrane and high Li migration and tLi+, whereas the aliphatic monomers containing ether groups were responsible for an efficient Li+ conducting path. The developed polymer electrolyte was obtained by a “structural self-assembly” and in-situ polymerization process by first blending the polymerizable monomers with PVDF-HFP dissolved in N-methyl-2-pyrrolidone (NMP) solution, with subsequent polymerization at 100 °C . Furthermore, a tLi+ of 0.9 was obtained, even after 800 h, which was crucial to suppressing dendrite formation, which was further confirmed by the Li stripping/plating cycle test performed using an SPE-based assembled cell. An impressive discharge capacity of 165 mAh·g−1 at 0.05C was also obtained, which slightly differs from the cathode’s theoretical specific capacity value of 170 mAh·g−1. Good cycling stability after 200 cycles was also achieved, with 95.5% retention, as well as nearly 100% coulombic efficiency. This promising work provides a simplified process suitable for future structural industrial applications.After soaking with plasticizer, the resultant porous and flexible-yet-stiff membrane delivered a tensile strength of 10.5 MPa and high ionic conductivity of 2.3 × 10−4 S·cm−1 at 25 °C and superior 0.93 tLi+, which delivered an optimal initial discharge capacity of 133 mAh·g−1 (1.0 C rate) for the respective Li/LiFePO4 cell.Another approach, based on this technique, used a Li (4-styrenesulfonyl) (trifluoromethanesulfonyl)imide monomer to develop highly crosslinked matrices with a superior electrochemical capacity . UV-induMoreover, apart from good rate capacity, the cell also exhibited superior cycling performance by keeping 83% of its retaining capacity after 400 cycles, with almost 100% coulombic efficiency.+ [3sp boron moiety (-B(O-)4). The developed poly  polymer (Li-PSB) was mixed with polybenzimidazole (PBI) binder, constituted by imidazole and aromatic groups, which enabled Li solvation and rigidity of the membrane, respectively. The fabricated membranes were subsequently immersed in EC/DEC electrolyte to produce a GPE, displaying enhanced fire-retardancy properties, good ionic conductivity at RT, and a high transference number . The incorporation of rigid aromaticity derived from the PBI binder resulted in a superior mechanical strength of 21.1 MPa. The electrochemical performance of the prepared Li/LFP resulting cell was also evaluated and a specific capacity of 131.8 mAh·g−1 (0.1 C rate) was found, as well as a 76.1% retaining capacity after 200 cycles, with nearly 100% coulombic efficiency. In this work, the inventors compared the developed GPE to a commercial separator and showed that their membrane presents superior benefits for fire retardancy, dendrite suppression, and higher ionic conductivity.Borate-based polymers have also been explored as potential electrolytes, due to boron’s low interaction with Li+ ,131,132.+ , a condu+ themselves, and the latter, despite being ionic insulators, offer a favorable environment for ion transport in a polymer matrix. The incorporation of inactive fillers enhances the free Li+ mobility and suppresses the polymer crystallization.When the design of polymer electrolytes alone is not enough to achieve the desired ionic conductivity, different strategies can be approached, including the incorporation of inorganic fillers into the SPE matrix. These fillers are normally divided into two categories: active fillers and inactive  fillers. The former are capable of conducting ions such Li2O3), achieving a significant enhancement of the mechanical stability of the composite. However, ionic conductivity improvements by adding Al2O3 particles into a PEO-based matrix were first proposed by Wieczorek et al. [The first study focused on the incorporation of inorganic (inactive) filler into a polymer electrolyte was in 1982, by Weston et al. . The autk et al.  in 1989.k et al. ,137,138.2) nanoceramic filler. The incorporation of TiO2 resulted in an improvement of the ionic conductivity , thermal stability (up to 350 °C), and mechanical strength (8.4 MPa). The TiO2 SPE also exhibited large tLi+ (0.53) as well as an extended electrochemical window of stability (5.4 V vs. Li/Li+).For example, Sasikumar et al.  reported2) as the filler and PEGDA as the crosslinker , a wider electrochemical window of stability (5.2 V vs. Li/Li+), and better ability to suppress dendrite growth.The combination of the mechanical rigidity of SiO2 fillers, succinonitrile as a plasticizer, and a cellulose membrane framework. The SPE membrane exhibited improved ionic conductivity , large tLi+ (0.8), and a high potential window (5.0 V vs. Li/Li+).Xu et al.  develope−3 S·cm−1 at 30 °C), excellent electrochemical stability up to 5.5 V vs. Li/Li+, and good thermal stability. A wide range of other inorganic inactive fillers can be found in the literature as potential reinforcements for SPE membranes, including cerium dioxide (CeO2) [2) [3N4) [2O5) [3N4) [In another recent study, a boron nitride (BN)-based SPE was developed . The croe (CeO2) , molybdeeO2) [2) , silicon2) [3N4) , niobium4) [2O5) , graphit5) [3N4) , quantum5) [3N4) , nitroge5) [3N4) , and alu5) [3N4) , among o+ ions. The most used are garnet-type  and its derivatives, such as tantalum-doped lithium lanthanum zirconate oxide (LLZTO)), NASICON-type , among others), and perovskite-type (such as lithium lanthanum titanate (LLTO)) [0.8 Co0.1 Mn0.1 (OH)2 (NCN811), and the electrolyte, an LLZO-reinforced PVDF/PAN/LiTFSI polymer electrolyte matrix (−4 S·cm−1), high tLi+ (0.84), and a broader electrochemical window (5.04 V vs. Li/Li+). A CR2032 coin cell containing Li/NCM811 achieved a capacity retention of 89.8% after 300 cycles at 1C and RT. Active fillers are more effective at boosting the electrochemical performance of SPEs since they can conduct Li (LLTO)) ,152,153. (LLTO))  developee matrix . The SPE−3 S·cm−1 at RT) and a remarkable tLi+ (0.85). Recently, a new type of Li-ion conductor oxide with trivalent gallium metal (LLGO) was also studied as an active filler for incorporation into SPEs. [−4 S·cm−1 at 60 °C) and a tLi+ as high as 0.69.Jin et al.  reportedto SPEs.  Using LL−4–10−3 S·cm−1, a wide electrochemical window, and enhanced thermal stability, they also show some drawbacks. For example, NASICON-type and perovskite-type reinforced electrolytes can be easily reduced by Li metal, and garnet-type reinforced electrolytes are unstable under air and easily react with water and carbon dioxide, generating lithium carbonate (Li2CO3) and lithium hydroxide (LiOH) [Although active fillers possess an ionic conductivity in the range of 10e (LiOH) .+-containing ionic liquid. The Co-MOF-reinforced PEO/LiTFSI SPE membrane showed high ionic conductivity , wide electrochemical stability (5.2 V vs. Li/Li+), and high tLi+ (0.5). In another attempt, Wu et al. [−5 S·cm−1 at 30 °C) and high tLi+ (0.75). The Ce-MOF SPE exhibited superior cycling stability, with a capacity of 120 mAh·g−1 after 3800 cycles when tested at 0.5 C, 60 °C, for a Li/LiFePO4 cell. Recently, metal–organic frameworks (MOF) consisting of metal nodes and organic ligands have attracted significant interest as fillers in SPEs. The high specific surface area of MOFs combined with their highly ordered crystal structures and porosity make these materials suitable for adsorbing liquid electrolytes and regulating ion flux. Furthermore, the organic–inorganic hybrid nature of MOFs can improve compatibility with SPEs . Zhang eu et al.  incorpor−3 S·cm−1, at 30 °C), a wide electrochemical window (5.25 V vs. Li/Li+), and a high tLi+ (0.61). It also exhibited good cycle stability in symmetric battery, with 98.6% capacity retention after 700 cycles (tested at 1C at RT).In addition, Zhang et al.  preparedSince the application of a liquid electrolyte is detrimental from a mechanical point of view, one of the main applications of SPEs/GPEs is their employment in structural batteries. These multifunctional devices are classified as a class of mass-less structural composites with high-density energy storage systems with enhanced mechanical properties ,174. In A structural battery can be produced using two main approaches: (i) through the incorporation of the functionalities into a component by, for example, making a laminated composite structure, and (ii) through the transformation of each component in a multifunctional material. In the first case, the electrolyte is capable of mechanical load transfer and is called a structural battery electrolyte (SBE). In the second case, the approach relies on an assembly of different constituents rather than a device with multifunctional components .−1), and great Li+ insertion capacity (280 mAh·g−1) [−1. In 2010, Eksted et al. [4 fiber weave as a cathode. Unfortunately, the mechanical properties attained were unacceptable and no electrochemical data were reported.CFs are used as anodes due to their high tensile strength (2–4 GPa), high electrical conductivity (4.9 S·cmmAh·g−1) ,177,178.mAh·g−1) ,148,179.mAh·g−1) . Howeverd et al.  develope−1) and withstand mechanical load (shear stiffness between 0.1 and 1 GPa) [Therefore, the challenge relies on the development of a safe solid/gel electrolyte able to efficiently conduct ions up to ~1 mS·cm mS·cm−1 d 1 GPa) . Anotherd 1 GPa) . Each cod 1 GPa) .2) as the cathode. The final assessment showed good mechanical properties with a 3.1 GPa tensile modulus but low specific energy  [+ salts was used as an electrolyte with an ionic conductivity of 2 × 10−4 S·cm−1 and a Young’s modulus of 0.5 GPa [−1 at 0.1 C and mechanical strength of 213 MPa. In this case, a graphitic anode and a CF coated LFP cathode were used to serve as current collectors.Other interesting examples reported the multifunctional mechanical properties of structural batteries. For instance, in 2009, Liu et al.  establisge rate) ,192,193.ge rate) ,194. A p 0.5 GPa ,193. MoyThere is still ample room available for optimizations in this field, especially concerning the mechanical point of view of a structural battery as well as the necessary characterizations.−3 S·cm−1; (ii) improved tLi+, as it favors the reduction of polarization and suppression Li dendrite growth; (iii) optimized interfaces between electrodes and electrolytes and an understanding of their interfacial behavior; (iv) enhanced electrochemical and thermal stabilities of SPEs to boost their lifespan; (v) low-cost preparation techniques for SPE as well as the sustainability of their polymer batteries for commercial applications; and (vi) mechanical stability for polymer electrolytes. The electrolyte must not be brittle, should be flexible and elastic, and should also stand the stress conditions during their lifespan. Despite the challenges outlined above, SPEs have been broadly recognized as one of the core directions to enhance the performance and safety of LIB. Bearing in mind the intense research and industrial interest in developing SPE and solid-state batteries, it is entirely reasonable to expect significant advances in the field to continue to occur.In recent years, significant research and progress in the field of solid-polymer electrolytes have been made. In this review, the recent advancements and research performed on the design of solid- and gel-polymer electrolytes were summarized, as well as composite polymer electrolytes, focusing their application on LIBs. Compared to liquid electrolytes, SPEs have the potential to offer more balanced overall battery performance in terms of their nonflammability, mechanical properties, ionic conductivity, safety, and thermal stability. The current limitations to SPE design were also outlined. Although solid-polymer electrolytes have shown great potential, challenges still need to be addressed to fulfil the optimized performance for practical applications. Strategies such as polymer blending, copolymerization, polymer crosslinking, and the development of single-ion polymer electrolytes, consisting of tethering the Li counter ion moieties to the polymer backbone, have been explored, aiming at optimizing SPE. Some key factors to be considered when designing SPE include (i) enhanced ionic conductivity at room temperature—it is still fairly challenging to achieve solvent-free polymer electrolytes with an ionic conductivity higher than 10


Candidatus Brocadia,” “Candidatus Kuenenia,” “Candidatus Scalindua,” “Candidatus Anammoxoglobus,” and “Candidatus Jettenia” were detected. Several operational taxonomic units (OTUs) from this study shared low sequence identities to the 16S rRNA gene of the known anammox bacteria, suggesting that they might be representing putative novel anammox bacteria. A quantitative PCR analysis of anammox-specific 16S rRNA gene confirmed that the abundance of anammox bacteria ranged from 1.60 × 104 to 1.20 × 107 copies L–1. Nitrate was a key environmental factor defining the geographical distribution of the anammox bacterial community in the hot spring ecosystem. Dissolved inorganic carbon had a significant influence on anammox bacterial biodiversity. Our findings for the first time revealed that the diverse anammox bacteria, including putative novel anammox bacterial candidates, were present in Conghua hot spring, which extended the existence of anammox bacteria to the hot springs in China and expands our knowledge of the biogeography of anammox bacteria. This work filled up the research lacuna of anammox bacteria in Chinese hot spring habitat and would guide for enrichment strategies of anammox bacteria of Conghua hot springs.Anaerobic ammonium oxidation (anammox) is an important process of the nitrogen cycle, and the anammox bacteria have been studied in a wide variety of environments. However, the distribution, diversity, and abundance of anammox bacteria in hot springs remain enigmatic. In this study, the anammox process was firstly investigated in hot springs of Conghua, China. Anammox-like bacterial sequences that closely affiliated to “ These hot spring water samples were collected during July 2018. Following collection, 20 L of each sample was filtered through 0.22 μm membrane filters  with a 150 mm diameter. Biomass on the filter was then transferred to a sterilized 50 mL round-bottom centrifuge tube  and stored with dry ice for further DNA extraction. The filtrates were collected in sterilized 250 mL white plastic bottles  with 58 mm in diameter and 103 mm in height, transported to the laboratory, and stored at −20°C for subsequent analysis of physicochemical properties.Six hot spring samples were collected from three stations: LiuXi River Left , LiuXi River Right , and GuanWeiHui  of Conghua, Guangdong province in China . Originsin situ using a portable multimeter . Nutrients in the water samples were analyzed according to standard procedures  and 1.0% agarose-gel electrophoresis. The 16S rRNA gene of anammox bacteria was amplified using a nested PCR approach with first-step primer pair PLA46F (5′-GGATTAGGCATGCAAGTC-3′)/1390R (5′-GACGGGCGGTGTGTACAA-3′)  according to the manufacturer’s protocols. DNA quantity and quality were checked using a NanoDropACAA-3′)  and secoACAA-3′) . PCR rea2O to a total volume of 20 μL. A standard plasmid carrying 16S rRNA gene fragment was generated by amplifying them from the extracted DNA sample and cloning into pEASY-T1 Vector . The concentration of plasmid DNA was determined on a NanoDrop® ND-2000 spectrophotometer , and the copy number of the target gene was calculated directly from the concentration of the extracted plasmid DNA. A significant linear relationship of 16S rRNA gene amplification (R2 = 0.9966) was obtained between the log 10 values of the standard plasmid DNA concentration (2.00 × 10–2.00 × 107 copies L–1) and the associated threshold cycles (Ct). Melting curves and gel electrophoresis were performed to confirm the specificity of the qPCR amplification. In all experiments, blank controls containing no template DNA were performed under the same qPCR procedure to ensure no contamination in our PCR system and the reliability of the results.The abundance of the 16S rRNA gene of anammox bacteria was determined in triplicate on a Bio-Rad iQ5 thermal cycler (Bio-Rad Laboratories) using primer sets AMX-808F (5′-ARCYGTAAACGATGGGCACTAA-3′)/AMX1040R (5′-CAGCCATGCAACACCTGTRATA-3′)  with a t1 with the vegan package. UpSetR .Analysis of raw Illumina fastq files was performed using Quantitative Insights Into Microbial Ecology 2  . DADA 2 . UpSetR  was usedThe basic physicochemical properties of hot spring samples collected from Conghua are shown in After quality control, a total of 181,802 high-quality anammox bacterial 16S rRNA gene sequences were obtained ranging between 13,884 and 57,231 sequences per sample , which wCandidatus Brocadia” (47.53%), “Candidatus Kuenenia” (0.134%), “Candidatus Anammoxoglobus” (0.896%), “Candidatus Jettenia” (0.133%), and “Candidatus Scalindua” (0.005%). The remaining unknown anammox bacterial OTUs accounted for 51.302%, which indicated that many putative novel anammox bacteria existed in the hot spring of Conghua. “Candidatus Brocadia” was the dominant genus in samples GWH2, GWH3, LXRL1, and LXRR2, while the putative novel anammox bacteria were dominant in samples GWH1 and LXRR1. “Candidatus Jettenia” was detected in GWH2, GWH3, and LXRR2; however, “Candidatus Kuenenia” and “Candidatus Scalindua” were only observed in LXRR2 and GWH3, respectively. The community structure of anammox bacteria in hot springs at the genus level is depicted in Based on the phylogenetic analysis, anammox-like bacteria that were closely affiliated to five known anammox bacterial genera were detected in the hot springs of Conghua. Among the 109 anammox bacterial OTUs in the collected samples, 65 OTUs were affiliated with “Brocadia clade, eighteen OTUs were retrieved from the samples. OTU 17 and OTU 25 were assigned to the “Candidatus Kuenenia” and “Candidatus Anammoxoglobus” clades, respectively, and ten OTUs belonged to the putative novel anammox genera, which means that genes of “Candidatus Brocadia” had the highest diversity, and “Candidatus Kuenenia” and “Candidatus Anammoxoglobus” had a lower diversity. BLASTN analyses revealed that the retrieved anammox bacterial 16S rRNA gene sequences in the hot spring of Conghua were also closely affiliated with sequences detected in paddy field soil, aquatic ecosystem, reactor, and sediment from the rhizosphere, wetland, riparian, estuarine, freshwater lake, and sea with a 92.65–100% sequence similarity. The anammox bacterial distribution pattern was verified at the OTU level. A phylogenetic tree  was cons4 to 1.20 × 107 copies L–1 (7 copies L–1) had the highest abundance of anammox bacteria, followed by sample GWH3 (8.51 × 105 copies L–1). The lowest abundance of anammox bacteria was observed in sample LXRL1 (1.60 × 104 copies L–1).Anammox bacterial 16S rRNA genes were used to estimate the abundance of anammox bacteria by the qPCR method. In this study, melting curve analyses confirmed that fluorescent signals were derived from the specific PCR products during qPCR quantification. The qPCR results with 97.43% amplification efficiency showed that the abundance of anammox bacteria ranged from 1.60 × 10pies L–1 . Sample CCA was conducted to show the potential relationships between environmental variables and the microbial communities of anammox bacteria in hot spring samples . The envp < 0.01) and soluble reactive phosphorus (p < 0.05) (p < 0.05). Anammox bacterial OTUs illustrated significantly positive correlations with dissolved inorganic carbon (p < 0.05). In addition, total organic carbon had a highly significant positive correlation with total carbon (p < 0.01), and soluble reactive phosphorus had a significantly positive correlation with temperature (p < 0.05)  . Shannon < 0.05) .Candidatus Scalindua,” “Candidatus Brocadia,” “Candidatus Kuenenia,” “Candidatus Jettenia,” and “Candidatus Anammoxoglobus”), and “Candidatus Brocadia” was identified as the dominant genus. To date, the diversity of anammox bacteria has been studied in various ecosystems. A low diversity of anammox bacteria was reported in marine ecosystems, which was mainly restricted to “Candidatus Scalindua”  can be found below: NCBI SRA BioProject, accession no: W-JL, LL, and J-YJ jointly conceived the study. W-DX, Z-TL, and J-YZ performed the sample collection. LL designed the experiments. A-PL, Y-ZM, and M-ML did the experiments. LL, X-TZ, and J-YJ analyzed the culture-independent data. LL, J-YJ, MPNR, NS, and W-JL wrote the manuscript. W-JL gave the final approved of the manuscript to be published. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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To describe the short-term outcomes of transvaginal natural orifice transluminal endoscopic surgery (vNOTES) for uterosacral ligament suspension (USLS) in patients with severe prolapse.This was a retrospective study of patients with severe prolapse (≥ stage 3) who underwent vNOTES for USLS between May 2019 and July 2020. The Pelvic Organ Prolapse Quantification (POP-Q) score, Pelvic Organ Prolapse/Urinary Incontinence Sexual Questionnaire short form (PISQ-12) and Pelvic Floor Inventory-20 (PFDI-20) were used to evaluate physical prolapse and quality of life before and after vNOTES for USLS.A total of 35 patients were included. The mean operative duration was 111.7 ± 39.4 min. The mean blood loss was 67.9 ± 35.8 ml. Statistically significant differences were observed between before and after vNOTES USLS in Aa (+ 0.6 ± 1.7 versus − 2.9 ± 0.2), Ba (+ 1.9 ± 2.2 versus − 2.9 ± 0.3), C (+ 1.5 ± 2.2 versus − 6.9 ± 0.9), Ap (− 1.4 ± 1.0 versus − 3.0 ± 0.1) and Bp (− 1.1 ± 1.4 versus − 2.9 ± 0.1) . The mean pre- and postoperative PFDI-20 score was 19.9 ± 6.7 and 3.2 ± 5.4, respectively, and the mean pre- and postoperative PISQ-12 score was 24.8 ± 2.3 and 38.3 ± 4.1, respectively (P < 0.05 for both). During 1–13 months of follow-up, there were no cases of severe complications or recurrence.vNOTES for USLS may be a feasible technique to manage severe prolapse, with promising short-term efficacy and safety. Larger studies with more patients and longer follow-up periods should be performed to evaluate the long-term efficacy and safety profile of vNOTES for USLS. Pelvic organ prolapse (POP) is a common benign condition with a high risk of occurrence of 40–60% in elderly women  and a reMany procedures, such as laparoscopic procedures via an abdominal approach and transvaginal procedures, have been described for USLS. However, transvaginal USLS carries up to an 11% risk of ureteral injury, which is attributed to poor visibility , 7. UngeIn recent years, some scholars have reported the advantages of transvaginal natural orifice transluminal endoscopic surgery (vNOTES) for USLS, which include no incisional pain and a better cosmetic outcome than laparoscopy via the abdominal approach, as well as direct visualization of key structures, such as the ureters and rectum, which is not available with the traditional transvaginal approach , 13. At In this study, we performed USLS for POP using the vNOTES approach. Our objective was to describe the short-term outcomes of vNOTES for USLS in patients with severe apical prolapse.We retrospectively collected data on all cases of vNOTES for USLS at the Obstetrics and Gynecology Hospital of Fudan University between May 2019 and July 2020. The inclusion criteria were as follows: (1) age 25–79 years; (2) severe apical prolapse (≥ stage 3); (3) desire for preservation of coital function; (4) first surgical treatment for POP; and (5) refusal of mesh implantation. The exclusion criteria were as follows: (1) inability to tolerate surgery; (2) coagulation dysfunction; (3) severe vaginal ulcers; (4) history of severe adhesions, a fixed uterus or strong pelvic adhesions noted on pelvic examination; (5) inability to tolerate the Trendelenburg position; and (6) suspicion of gynaecological malignancy. This retrospective study was approved by our institutional review board before any data were collected (no. 2019–32).We extracted the following information from medical records: patient demographics  and perioperative outcomes. Perioperative data included the operative duration, blood loss, intraoperative complications (transfusion or injury), postoperative complications , length of postoperative hospital stay, and hospitalization costs. We also assessed the change in physical prolapse with Pelvic Organ Prolapse Quantification (POP-Q). Two validated questionnaires, the Pelvic Organ Prolapse/Urinary Incontinence Sexual Questionnaire short form (PISQ-12) and Pelvic Floor Inventory-20 (PFDI-20), were completed before and at least 3 months after vNOTES for USLS to assess the impact on quality of life. Postoperative follow-up visits were scheduled at 1 month, 3 months, 6 months, 1 year and 2 years after surgery. Any POP-Q score greater than or equal to − 1 cm was defined as recurrence .All participants underwent surgery with general anaesthesia and standard operative care. Patients treated with hysterectomy received cefuroxime preoperatively. A single-port device with four trocars  and nonabsorbable or delayed absorbable sutures  were used.This surgical procedure was originally described by Lowenstein et al. in 2019 . In thisThe single-port platform was placed from the vaginal orifice to the pelvic cavity through the posterior fornix. In these cases, apical support was achieved by suturing the USLs, shortening the ligaments (as mentioned above) Fig. i and reiIn patients who desire preservation of the uterus without a requirement for fertility, part of the cervix was excised according to the degree of cervical extension Fig. iii, and Anterior or posterior colporrhaphy is necessary when the anterior or posterior compartment reaches stage II prolapse. Perineal body repair was performed at the discretion of the operating surgeon such that the width of the vaginal orifice was more than that of 3 fingers at the end of the procedure. Anti-incontinence surgery was allowed according to the preoperative presence of stress urinary incontinence (SUI). The placement of a tension-free midurethral sling (MUS) was performed in patients who were willing to undergo mesh placement. If not, urethral folding was performed. Gauze was inserted into the vagina for compression.After surgery, routine postoperative care was provided. Cefuroxime was administered once postoperatively in patients treated with hysterectomy. All patients were then followed clinically; the longest follow-up duration was 13 months.Data collection and statistical analyses were performed using IBM SPSS Statistics 24.0 software . All variables are presented as the mean and standard deviation (SD) or n and percentage (%). Continuous variables were compared by Student’s t test. A P value < 0.05 was considered statistically significant.In total, 35 patients underwent vNOTES for USLS; in one patient, vNOTES sacrocolpopexy was planned, but the patient was converted to vNOTES for USLS because of a suspected malignant lesion on the surface of the colon. The patient characteristics are shown in Table Table Escherichia coli, and was cured after antibacterial treatment. One patient (2.9%) developed postoperative urinary retention; this patient underwent catheterization for urine drainage and was cured after 3 days of acupuncture treatment. One patient (2.9%) suffered from constipation, which was relieved with a glycerine enema. There were no cases of de novo SUI, deep vein thrombosis, persistent pain, haematoma or dyspareunia as postoperative complications. The mean follow-up duration was 3.9 ± 3.8 (1–13) months. No patients had obvious recurrence.There were no cases of intraoperative blood transfusion or injury Table . In totaTables In the present study, vNOTES for USLS resulted in marked improvement in both anatomical prolapse and quality of life without cases of conversion or serious peri- or postoperative complications. These results suggest that vNOTES for USLS may be a feasible technique for treating severe POP.USLS is a classic surgical method for the treatment of middle compartment POP. vNOTES has the advantages of two traditional surgical approaches, including good exposure, no abdominal wound, and more accurate suture placement after confirmation of ischial spine localization. Studies have shown that the intermediate portion of the USL at the level of the ischial spine is the strongest. Management of the anterior vaginal wall is very important. First, anterior colporrhaphy was performed when the anterior compartment reached stage II prolapse. Lee et al. reported that concomitant anterior colporrhaphy at the time of USLS seemed to reduce the recurrence of anterior vaginal wall prolapse . Second,POP is often accompanied by SUI, which is easy to identify when patients have symptoms. In this study, five patients were diagnosed with SUI by preoperative symptoms and urodynamic examination results, and all of them underwent concomitant anti-incontinence surgery. To date, there have been no cases of de novo SUI during follow-up. This is consistent with other reports in the literature; when POP is combined with SUI, prolapse repair surgery combined with anti-incontinence surgery can reduce the risk of postoperative de novo SUI , 18. OccThe results of this study suggest that sexual activity was restored 3 months after surgery. However, only nine of the 17 patients recovered their sexual life. There were many factors affecting the sex lives, such as age, surgery, spouse, psychological factors, and et al. In contrast with women in Western countries, Chinese women experience a rapid decrease in sexual need with age, especially after the age of 50 .At our hospital , vNOTES This study has several limitations. First, the follow-up duration was relatively short, and the sample size was limited. Another, this was an observational study with no control group and there lied in the inherent weaknesses of retrospective studies. However, to our knowledge, there are few articles to describe the surgical outcomes of vNOTES for USLS, which may provide some references.vNOTES for USLS, with promising short-term efficacy and safety, may be a feasible technique for the treatment of severe prolapse. Hence, additional studies with a larger number of patients and a longer follow-up period should be conducted.