Although autografts are not feasible in patients with extensive burn wounds, allografts and xenografts can be used for temporary coverage. In this systematic review and meta-analysis, we compared the outcomes of xenografts and the standard treatment of burn wounds.International online databases were searched for English articles comparing xenografts with routine treatment in the burn patients. The random-effects model was used to estimate standardized mean differences (SMD) or odds ratios (OR) with a 95% confidence interval (CI).From a total of 7144 records, 14 studies were included in our review after screening by title and abstracts followed by full-texts. No significant difference in hospital stays was found between the mammalian xenografts and control groups (SMD [95% CI] = − 0.18 [− 0.54–0.18]). The mean number of dressing changes was significantly lower in both mammalian xenografts compared to the controls (SMD [95% CI] = − 1.01 [− 1.61–− 0.41]) and fish xenografts compared to controls (SMD [95% CI] = − 6.16 [− 7.65–− 4.66]). In the fish xenografts, re-epithelialization time was significantly lower compared to controls (SMD [95% CI] = − 1.18 [− 2.23–− 0.14]).Xenografts showed a significantly lower number of dressing changes and fish xenografts showed significant benefit in re-epithelialization compared to routine treatment. The beneficial results of xenografts suggest further research in the use of different types of xenografts in patients with extensive burn.The online version contains supplementary material available at 10.1186/s40001-023-01505-9. Burn injuries result in inflammation and metabolic disturbances, leading to shock, multi-organ failure, and considerable morbidity and mortality. Burn injuries are caused by exposure to thermal, chemical, and electrical sources and radiation leading to tissue damage by different mechanisms. Moreover, the management and outcome of burn injuries greatly result from the depth and size of the wound .Deep burns are more likely to have complications, such as wound infection, sepsis, shock, and scarring tissue contraction . Wound iSilver sulfadiazine (SSD) is an antimicrobial topical agent with a low risk of bacterial resistance, adverse effects, and toxicity. It has been used for treating chronic and burn wounds for a long time but the abundant number of dressing changes and the resultant pain propose the necessity of better treatment .The gold standard treatment of deep partial thickness and full thickness burn is early excision and skin grafts , 7. ContBiological skin substitutes must have adequate strength, flexibility, adhesion to the wound, good aesthetic results, and remodeling ability to provide an optimal wound repair and healing. Moreover, their safety in terms of risk of infectious disease transmission, microorganism penetration, toxicity, oncogenicity, and allergenicity is concerning . A PorciAnother commonly used xenograft is derived from fish which has been reported in some cases of burn wound treatment and neovaginoplasty , 14. Nilhttps://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022373748).This systematic review and meta-analysis were designed and performed in accordance with “Preferred Reporting Items for Systematic Reviews and Meta-Analyses” (PRISMA) . RegistrHuman or animal in vivo investigation of the effects of xenografts in burn wounds was determined as the fundamental inclusion criterion.(P) Population: Burn patients.(I) Intervention: Xenograft/Animal transplant/Animal graft.(C) Comparison: Open.(O) Outcomes: Open.(S) Study: Clinical trials and animal studies.According to these PICOS questions, we designed the following clinical question: in burn patients, what are the outcomes of the use of xenografts compared to routine treatment? Exclusion criteria included studies before the year 2000, studies other than trials and animal studies, and the studies that used genetically modified or not skin xenografts. Articles without full-text or not in English were excluded as well.We systematically searched PubMed, Cochrane Library, Scopus, and Web of Science without any filters or limitations until October 28, 2022. Keywords were Xeno* AND burn* with other related search terms shown in Additional file Two independent reviewers (R.S. and H.B.) carried out the selection of studies. After removing the duplicates and screening the articles based on their title and abstracts, full-text of studies were retrieved to select the relevant studies according to the inclusion and exclusion criteria. Any disagreement during the article selection was resolved thanks to one independent investigator (R.I.).The data extraction process was performed by two independent researchers (H.B. and R.I.). The extraction tables included the name of the authors, country of origin, year of publication, study design, participant characteristics, including number, age, and TBSA, type of xenograft, detailed information about the surgeries that were operated on intervention and control groups, outcomes and complications in both groups. In addition, the review authors looked for the sources of funding for the studies included in the review.Two independent reviewers (H.B. and R.I.) evaluated the risk of bias in each article by means of "Cochrane Handbook for systematic reviews of interventions, version 5.1.0" . We evalP < 0.05 for all analyses. Forest plots were (JUSTIFICACIÓN DEL TEXTO) created to illustrate the effects in the meta-analysis of the global estimation. The heterogeneity of ≤ 25% was considered as low, 26–75% as moderate, and > 75% as high [Standard mean difference (SMD) with 95% confidence intervals (CI) was used for comparing xenografts with other treatments of burns. Pairwise meta-analyses were conducted using RevMan software . Statistical significance was defined as  as high . Due to n = 1671) and the irrelevant articles based on their title and abstracts, 64 studies were screened. After full-text screening and implementing inclusion and exclusion criteria, 14 studies were included . In addition, Zajicek et al. [In the Feng et al. trial , the timk et al.  found a k et al.  found a In Zuo et al.  study, tIn the Zajicek et al. study , completKarlsson et al.  reportedIn the study conducted by Feng et al. , the scaP = 0.33; I2 = 54%; Fig. P = 0.331). In Hosseini et al. [p = 0.001), respectively. In Hosseini et al. study in 2009 [p = 0.11). In line, Tuleubayev et al. [No significant difference between hospital stays was found in the meta-analysis of 4 studies , respectively. Finally, in Hosseini et al. study in 2009 [P = 0.0005).In the meta-analysis of 3 studies , 28, 29,P = 0.001).Two studies by Hosseini et al. compared mortality between the xenograft group and controls; in the Hosseini et al.  trial inP = 0.03; I2 = 90%; Fig. p = 0.001). In the Stone et al. study in 2018 [P < 0.005). The contraction rates were reported as the percentage of original size and a significant reduction in original wound size at day 14 was observed for the FSG when compared to FBD .Day to re-epithelialization was significantly lower in fish xenografts versus controls in the meta-analysis .The number of dressing changes was significantly lower in fish xenografts compared to controls in the meta-analysis  topical silver agents, (2) biological dressings, including amniotic membrane, allografts, xenografts, and bioengineered dressings, (3) enzymatic debridement, and 4) surgery  surgery , 7. AlthIn a study by Brown et al.  effectivBurn treatment takes up a large share of financial resources, especially with deep wounds, the treatment is very expensive and imposes a large financial burden on the health system . The numLength of hospital stay in burn patients is positively correlated with TBSA . MoreoveOur study had some limitations. First, since some studies had not reported sufficient data to perform secondary analysis, meta-analysis was not possible for all outcomes. Second, the control groups in studies were different  which can impact the findings of this study. Third, using non-randomized trials can impact the final findings by possible selection bias in individual studies. Finally, different TBSA and other baseline characteristics of patients emphasize the need for designing large clinical trials to better compare xenografts with other conventional treatments.In this study, we retrieved that xenografts showed a significantly lower number of dressing changes; the number of days to re-epithelialization showed significant reduction in fish xenografts compared to routine treatment. The beneficial results of xenografts suggest further research in the use of different types of them in burn patients who need a large amount of grafts.Name of the registry: PROSPERO.2.Unique Identifying number or registration ID: CRD42022373748.3.https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022373748Hyperlink to your specific registration (must be publicly accessible and will be checked): Additional file 1: Table S1. Search strategy and keywords. Table S2. Quality assessment of randomized clinical trials using cochrane tool of risk of bias assessment (RoB). Table S3. Quality assessment of non-randomized clinical trials using risk of bias in non-randomized studies – of interventions (ROBINS-I). Table S4. Quality assessment of non-randomized clinical trials using SYRCLE.


According to some studies, diet can be interaction with CRY1 polymorphism and may be related to obesity and the risk of cardiovascular diseases (CVD). So, this study examined the interaction between CRY1 polymorphism and AHEI on cardiovascular risk factors in overweight women and women with obesity.2). Dietary intake was evaluated by the use of a food frequency questionnaire (FFQ) with 147 items. The AHEI was calculated based on previous studies. Anthropometric and biochemical measurements were assessed and the bioelectrical impedance analysis method was used for body analysis. The rs2287161 was genotyped by the restriction fragment length polymorphism (PCR-RFLP) method. Objects were divided into three groups based on rs2287161 genotypes.This cross-sectional study was performed on 377 Iranian women with overweight and obesity aged 18–48, this study demonstrated that there was a significant interaction between CC genotype and adherence to AHEI on odds of hyper LDL , hypertension  and hyperglycemia .Our findings determined that the prevalence of the C allele was 51.9% and the G allele was 48.0%. The mean age and BMI were 36.6 This study indicated that adherence to AHEI can reduce the odds of hyper LDL, hypertension, and hyperglycemia in the CC genotype of rs2287161. In recent years, more than 1.9 billion adults aged 18 years and older were overweight and over 650 million adults were obese , 3. WorrAmong environmental factors, the diet has a crucial role in obesity and CVDs development , 11. In According to recent studies, genetics is an effective factor in the etiology of chronic diseases . CircadiPrevious studies propose the contribution of genetics, dietary, and environmental factors may play a significant role in the pathogenesis of obesity and CVDs , 26. AccNumerous studies found an inverse association between the higher score of AHEI cardiovascular risk factor and as mentioned, CRY1 rs10861688 is associated with cardiovascular disease , 32. As 2), not being pregnant, latescent, menopause, and no smoking and alcohol consumption. Moreover, we excluded subjects with a history of cardiovascular disease, cancer, diabetes, sustained hypertension, thyroid disease, acute or chronic infections, and liver and kidney disease. We also excluded subjects with following an unusual dietary routine, having significant weight fluctuations in the past 1 year, use of dietary supplements and weight loss medications, and participants whose conveyed daily energy intakes were 800 kcal/day or 4200 kcal/day.This cross-sectional study was carried out on 377 overweight women/ women with obesity who were referred to health centers in Tehran, Iran. Subjects were registered by the use of a simple sampling method. Inclusion criteria were age 18–48 years, being overweight or obese (body mass index (BMI) ≥ 25 kg/mBefore taking part in the study, each subject completed a written informed consent form all procedures involving human subjects were approved by the Ethics Commission of Tehran University of Medical Sciences (IR.TUMS.VCR.REC.1398.051). The research was done with the support of the Tehran University of Medical Sciences (Grant number: 99.3.212.50265).Data on age, education , marriage (single or married), occupation (employee or unemployed), economy , history of weight loss in previous years (yes or no), history of family obesity (yes or no) were collected by trained nutritionist and using a demographic questionnaire.Physical activity was assessed by the use of the generally validated International Physical Activity Questionnaire (IPAQ)  and selfParticipants’ usual dietary intake for any food consumed daily, weekly, or monthly during the past year was found using a 147-item semi-quantitative food frequency questionnaire (FFQ) with a skilled nutritionist. The validity and reliability of this questionnaire have been previously reported . PortionThe Alternative Healthy Eating Index (AHEI) contains 11 components including 1- fruits, 2- vegetables, 3- whole grains, 4- nuts and legumes, 5- long-chain omega-3 fatty acids, 6- polyunsaturated fatty acids, 7- sugar-sweetened beverages and fruit juice, 8- red and processed meats, 9- trans fatty acids, 10- sodium, 11- alcohol. We did not consider alcohol intake due to a lack of information. The consumption of fruits, vegetables, whole grains, nuts and legumes, long-chain omega-3 fatty acids, and polyunsaturated fatty acids were scored 10 and 0 in the highest and lowest intake, respectively. Also, the consumption of sugar-sweetened beverages and fruit juice, red and processed meats, trans fatty acids, and sodium were scored 10 and 0 in the lowest and highest consumption. Therefore, the AHEI score ranges from 0 (non-adherence) to 100 (perfect adherence) . Partici2 (m2).Weight was measured by using a digital scale  with a sensitivity of 0.1 kg. Height was measured by the use of a non-flexible tape measure and recorded to the nearest 0.1 cm. The measurement was performed while the participants were in a standing position, with light clothes and no shoes. Waist circumference (WC) and hip circumference (HC) were measured in the central point of the iliac crest and rib cage with flexible tape with an accuracy of 0.1 cm. The waist-to-hip ratio was calculated by dividing WC by HC. BMI was also calculated by use of the equation “weight (kg) / heightBody composition of subjects was evaluated by Body Composition Analyzer BC-418MA- In Body (United Kingdom). This Bioelectrical Impedance Analyzer (BIA) is considered to send a very weak electric current to assess the body impedance . We followed all of the following instructions for an accurate measurement. To avoid possible discrepancies in the measured values, participants were asked not to exercise vigorously, do not use any electrical devices, and not consume too much fluid or food before evaluating their body composition. Body composition analysis was performed while participants were fasting and urinating . Blood pressure.Blood pressure was measured after the participants had been at rest for 10 min. Hypertension was defined as systolic blood pressure ≥ 130 mm Hg and/or diastolic blood pressure ≥ 85 mm Hg .To assessment fasting serum glucose, insulin, and serum lipids, enzymes blood samples were assessed after 8–12 h of fasting at the Nutrition and Biochemistry Laboratory of the school of Nutritional and Dietetics at Tehran University of medical sciences. Fasting blood sugar (FBS) was measured on the day of blood accumulation by phenol-4-aminoanthyrine peroxidase (GOD / PAP) glucose oxidase. The concentration of serum triglycerides (TG) by use of the kits triacylglycerol  glycerol-3-phosphate oxidase method using phenol 4-Mynvanty Pirin peroxidase (GPOPAP) were assessed. Total cholesterol  levels were measured by the cholesterol oxidase Phenol 4-Aminoantipyrine Peroxidase (CHOD-PAP), and low-density lipoprotein (LDL) and high-density lipoprotein (HDL) was assessed by the direct method and immunoinhibition. Serum insulin concentrations were analyzed through the enzyme-linked immunosorbent assay (ELISA) method  . HypertrHomeostatic model assessment-insulin resistance (HOMA-IR), was determined based on the following equation: [fasting plasma glucose (mmol/l) × fasting plasma insulin (mIU/l)]/22.5 .All participants from whom deoxyribonucleic acid (DNA) samples were accessible, were evaluated to be genotyped for the rs2287161. Genomic DNA extraction from whole blood samples was performed using Mini Columns  based on the manufacturer’s protocol. The concentration and quality of the extracted DNA were assessed by the use of a NanoDrop ND-2000 spectrometer. The rs2287161  was genotyped by polymerase chain reaction-restricted length polymorphism (PCR–RFLP) technique. PCR applied the following primers: forward 5′-GGAACAGTGATTGGCTCTATCT − 3′; reverse 5′-GGTCCTCGGTCTCAAGAAG-3′. PCR reactions were done in a final volume of 20 µl include of 2 µl primers, 1 µl extracted DNA,7 µl distilled water, and 10 µl Taq DNA Polymerase Master Mix  with the next conditions in a DNA thermocycler: The DNA templates were denatured at 94° C for 4 min; amplification contained 35 cycles at 94 °C, 58 and 72 °C (each stage for 30 s), with a final extension at 72 °C for 7 min. Amplified DNA (10 µl) was mixed with 2 µl of the DRI restriction enzyme  at 37 ° C. To ensure the PCR process and amplification of the desired parts, PCR product electrophoresis was performed on the agarose gel. Fragments including three possible genotypes were then determined: uncut homozygous GG (107 bp), cut heterozygous GC , and cut homozygous CC (155 and 226 bp) .n = 122), group 2 with GC genotype (n = 148), and group 3 with GG genotype (n = 107). The binary logistic regression model was used to analyze potential interactions between rs2287161 genotype and quartiles of AHEI on odds of CVDs risk factors before and after adjustment for confounders . Also, the remaining method was used to control energy in the AHEI. Moreover, we calculated the percentage of cardiovascular risk factors across CC, GC, and GG genotypes by chi-square test. Data were analyzed using IBM SPSS version 23.0 . P < 0.05 was considered statistically significant.Normality distribution was analyzed by applying Kolmogorov–Smirnov’s test. Information of continue characteristics was shown as the mean ± SD and information of categorical characteristics was reported as a number and percentage. A comparison of continuous and categorical variables across the quartiles of AHEI or genotypes was done by the use of one-way analysis of variance (ANOVA) and chi-square, respectively. Also, we adjusted variables for confounders  by Analysis Of Covariance (ANCOVA). Genotypes were recorded based on risk allele: code 0 for GG, 1 for GC, and 2 for CC genotype. In order to examine the interactions between rs2287161 genotype and quartiles of AHEI on odds of CVDs risk factors, the participants were grouped based on CRY1 genotypes: group 1 with CC genotype  of age, weight, and BMI of participants were 36.67 n = 107), GC genotype (n = 148), and CC genotype (n = 122) (Table P = 0.04), fat mass index (FMI) (P = 0.05), high-density lipoprotein (HDL) (P = 0.03), alanine aminotransferase (ALT) (P = 0.05), pulse (P = 0.03), economic status (P = 0.007). Also, we obtained significant differences between genotypes for age (P = 0.04), physical activity (P = 0.04), fasting blood sugar (FBS) (P = 0.05), and education (P = 0.01) after adjustment for confounders .The whole number of 377 Iranian women were grouped according to rs2287161 genotypes and placed into three groups: GG genotype (P = 0.06) across rs2287161 genotypes. Also, after controlling for Energy intake across rs2287161 genotypes, there were significant differences in vegetables. No significant differences were observed for other variables , BMI (P = 0.01), fat free mass (FFM) (P = 0.03), skeletal muscle mass (SMM) (P = 0.03), waist circumference (WC) (P = 0.02), low-density lipoprotein (LDL) (P = 0.05), systolic blood pressure (SBP) (P = 0.001) and diastolic blood pressure (DBP) (P = 0.02) across quartiles of AHEI. Also, we observed a significant difference for age (P = 0.05) and physical activity (P = 0.04) after adjustment for confounding variables . All subjects were assessed across quartiles of AHEI Table . We founBy use of the binary logistic regression model analysis, the interaction between CRY1 polymorphism (rs2287161) and quartiles of AHEI on CVD risk factors was examined. In the models, we included a risk allele genotype (CC genotype), quartiles of AHEI, and interaction between CRY1 polymorphism (rs2287161) and quartiles of AHEI. In the crude model, there was a significant interaction between rs2287161 genotypes and adherence to AHEI on odds of hyper LDL  and hypertension . After adjusting for age, energy intake, physical activity, FFM, and socioeconomic status, a significant interaction was found between rs2287161 genotypes and adherence to AHEI hyper LDL , hypertension  and hyperglycemia . There was no significant interaction between rs2287161 genotypes and quartiles of AHEI on other CVD risk factors. Also, we found that adherence to AHEI can decrease the risk of hypo HDL, central obesity, and hypercholesterolemia across CC genotype but their interactions were not significant (Fig. Additionally, 31.7% of participants in the first quartile, 19.5% of participants in the second quartile, 19.5% of participants in third the quartile, and 29.3% of participants in the last quartile had CC genotype.This cross-sectional study examined the interaction between the AHEI and CRY1 gene polymorphism on CVDs risk factors in overweight women and women with obesity. The results showed that a negative and strong association was found between adherence to AHEI and the odds of hyper LDL, hypertension, and hyperglycemia across CC genotype. Generally, adherence to AHEI modified the association of the rs2287161 genotypes with the odds of CVDs risk factors such as hyper LDL, hyperglycemia, hypertension, hypo HDL, central obesity, and hypercholesterolemia in the CC genotype.In the study, we found that the CC genotype had mean higher FBS, ALT, BMI, and FMI and lower HDL and pulse with statistically significant differences. Several studies have presented the crucial role of the CRY gene via the inflection on several chronic diseases. A large sample study that is part of a national health survey in the adult-aged ≥ 30 years showed that the CRY1 genetic variants have a role in elevated blood pressure and high triglyceride . FurtherA finding of this study indicated that adherence to AHEI in CC genotype decreased the hyper LDL, hypertension, and hyperglycemia with statistically significant differences and reduce hypo HDL, central obesity, and hypercholesterolemia without statistically significant differences. A case cohort study found that tendency to the negative association between adherence to AHEI and diabetes in European populations . PreviouHowever, the interaction between diet and genes is rarely considered, thus, we investigated the interaction between the AHEI and CRY1polymorphism on cardiovascular risk factors. Although several studies have assessed the interaction of CRY and diet, such as obesity, diabetes, and insulin resistance, no research has been shown to evaluate the interaction between AHEI and CRY1 polymorphism. A cohort study in the Mediterranean and North American indicated that the CRY1 polymorphism had a significant interaction with carbohydrate intake for HOMA-IR and fasting insulin . A studyIt seems that the mechanism of this interaction between the CC genotype and adherence to AHEI with a reduced risk of CVDs risk factor might be due to the contribution of CRY1 in the regulation of steroidogenesis and gluconeogenesis . CRY1 deThe strength of this study is that it is the first research to estimate the interaction between CRY1 polymorphism and AHEI on the odds of CVDs risk factors in participants, and it was a community-based study. However, our study has some limitations. The cross-sectional design of the study, cannot determine the mechanism of the association between AHEI and the rs2287161 genotype. Also despite controlling several confounding variables in our study, the possible influence of remaining confounders should be considered.The results of this study indicate that adherence to AHEI can reduce the odds of hyper LDL, hypertension, and hyperglycemia in the CC genotype. However, the mechanism of interaction between AHEI and CC genotypes is not clearly understood. The results of our study suggest that dietary intake, gene types, and their interactions must be considered in CVDs risk factors evaluation.


To compare and analyze the mucosal metabolites and mucosal microbiota of different parts of colon in patients with IBS.A total of 10 patients with IBS-D and six healthy controls (HC) were enrolled. All enrolled participants underwent two biopsies of the ileocecal and sigmoid colon during colonoscopy. Metabolomic profiling of one piece of tissue was conducted using desorption electrospray ionization-mass spectrometry (DESI-MS), and the gut flora of the other piece was examined using 16S rRNA sequencing. The metabolic profiles and flora of the ileocecal and sigmoid colonic mucosa in each group were further analyzed in this study.p = 0.40, p = 0.22). However, in the HC group, microbiome analysis of the ileocecal mucosa showed lower values for Chao 1 and Shannon indices than those of the sigmoid colon mucosa . (3) Compared with the HC group, 1,113 metabolic signal peaks were upregulated, whereas 594 metabolites were downregulated in the IBS-D samples. Moreover, the PCA of the metabolites showed significant separation between the IBS-D and HC groups. (4) Chao1 expression was significantly higher in the mucosal microbiota with IBS-D than in the HC (p = 0.03). The Shannon index was lower in IBS-D, but the difference was not statistically significant (p = 0.53). PCoA revealed a significant difference in the microflora structure between the IBS-D and HC groups.(1) Principal components analysis (PCA) indicated that mucosal metabolites did not differ in different parts of the colon in either the IBS-D or HC groups. (2) In the mucosal microbiome analyses, no differences between the microbiota of the two parts of the colon were found by using Principal Co-ordinates Analysis (PCoA). In IBS group, comparing with sigmoid mucosa, the chao1 richness indice was higher and the Shannon index was lower in the ileocecal mucosa (The mucosal metabolic profile and mucosal flora structure of the colon were similar, despite different locations in IBS and healthy subjects. IBS had abnormal colonic mucosal metabolism and flora disturbances. Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder, with a prevalence of 11% , 3. ExplRecent metabonomics techniques examine small molecules in biological samples and detect subtle changes reflecting different physiological and pathological conditions, which have also been used to identify potential metabolic profiles for IBS. Recently, several metabonomics technologies, such as hydrogen nuclear magnetic resonance (H-NMR), liquid chromatography mass spectrometry (LC–MS), gas chromatography mass spectrometry (GC–MS), and high pressure liquid chromatography–tandem mass spectrometry (HPLC–MS), have revealed abnormal glucose, lipid, and amino acid metabolism in patients with IBS . These rGut microflora dysbiosis is a major mechanism underlying IBS . The colThe study applied DESI-MSI and 16S rRNA sequencing for ileocecal and sigmoid colon mucosa in IBS-D patients and healthy controls. This study aimed to compare the differences in mucosal metabolites and microbiota from different parts of the colon and to explore the characteristics of mucosal metabolism and microbiota in IBS-D.We performed a prospective study in IBS-D patients and healthy controls (HC). Ten adult patients with IBS-D who met the Rome IV criteria were recruited from the Department of Gastroenterology at our hospital between May 2022 and August 2022. The severity of IBS-D was measured using the IBS Symptom Severity Scale (IBS-SSS), which includes five items . Six healthy volunteers from the community participated in this study. Healthy subjects without organic intestinal disease or gastrointestinal symptoms were recruited as controls.The inclusion criteria for all enrolled subjects were as follows: age between 18 and 65 years; normal blood count; alanine aminotransferase (ALT), alkaline phosphatase (ALP), and serum creatinine within reference values; and normal thyroid function. Exclusion criteria included the following: history of polyps in 3 years; malignant tumors; history of major gastrointestinal surgery; diarrhea due to other medical illnesses ; chronic opioid or antidepressant use; pregnancy; and breastfeeding. None of the participants were allowed to take any prebiotics, probiotics, antibiotics or antidiarrheal medications within 4 weeks before recruitment.Colonoscopies were performed in both patients with IBS-D and healthy controls. All enrolled participants underwent two biopsies of both the ileocecal and sigmoid colons during colonoscopy. Two biopsy specimens from each part of the colon were used for DESI-MSI and 16S rRNA sequencing. The study protocol was approved by the Human Ethical Committee of the China-Japan Friendship Hospital, and all participants provided informed consent.The ileocecal and sigmoid specimens were snap-frozen in liquid nitrogen immediately after biopsy removal and stored at −80°C until further processing. One piece of the frozen samples from the same location was embedded in 5% sodium carboxymethyl cellulose (CMC). Frozen sections (20 μm thick) were stored at −80°C for DESI–MSI and 9 μm thickness for stained with hematoxylin and eosin (H&E). The other piece was examined using 16S rRNA sequencing for intestinal flora analysis.All MSI experiments were performed using a SYNAPT G2-Si HDMS DESI XS instrument  and a Harvard Apparatus Pump11 Elite. Glass slides containing 20 μm slices were subjected to DESI–MS imaging in the positive and negative ion modes over the mass range m/z 500–1,100 and 100–1,000. Then 1,000 peaks with the highest intensities were selected. The spray solvent for DESI was methanol/water in a ratio of 98:2, containing 200 ppb leucine encephalin, and injected at a rate of 3 μL/min. The parameter settings were as follows: capillary temperature, 150°C; nitrogen spray, 0.45 MPa; capillary voltage, 3.5 kV. Tissues were performed in constant velocity scan mode with a velocity of 100 μm/s and set at a spatial resolution of 50 μm to acquire DESI–MS images.p < 0.05), Student’s t-test] versus log2 (mean fold change) was plotted. The mass spectrometry peaks were annotated to known compounds based on the HMDBFour equal-area regions of interest (ROI) were selected on the mucosal layer of each DESI image and compared with the H&E staining images. EZinfo 3.0 (Waters) was used to further process the DESI-MS imaging data. Multivariate analyses were applied to the metabolite data. A principal component analysis (PCA) model served was used to identify potential differences between the two groups. Volcano scatter plots were used to identify differentiating metabolites, for which the fold change for each metabolite between IBS-D and healthy controls was calculated . Significance [−log10  and 806R (GGACTACNNGGG- TATCTAAT), the V3-4 regions of the bacterial 16S rRNA gene were amplified. Individual amplification products were purified and a sequencing library was constructed. Library quality was assessed on a Qubit@2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2,100 system, the library was sequenced on an Illumina NovaSeq platform, and 250 bp paired-end reads were generated, spliced, and filtered to obtain clean data. Operational taxonomic units (OTUs) clustering and species classification were performed. Visualization and comparison of intestinal flora were performed using a Principal Co-ordinates Analysis (PCoA) model with a permutation testing algorithm to detect microbiota variations between the two groups.t-tests were used for comparisons between the IBS-D and HC groups. Statistical significance was defined as p < 0.05.Statistical analyses were performed using the dedicated statistical software SPSS  and GraphPad Prism version 8.0. Student’s 2) and six healthy subjects . There were no significant differences in age or BMI between the patients and healthy subjects. The mean IBS-SSS in patients with IBS-D was 257.7. A total of 32 biopsy specimens were obtained from the 16 subjects. Specifically, 12 biopsies from healthy controls  and 20 from patients with IBS-D  were collected.In total, our study comprised 10 patients with IBS-D   . PCoA coctively) .p = 0.40, p = 0.22)  . PCoA re = 0.22) .p < 0.05 between the two groups in both the positive and negative ion modes. When compared with the HC group, the higher detected metabolites of the colon mucosa in patients with IBS-D are represented by red spots, lower ones are represented by green spots, and black spots indicate no significant differences between the two groups. Compared to the HC group, we found that 401 metabolite peaks of IBS-D were upregulated and 304 peaks of IBS-D were downregulated in the positive ion mode  and Lipid Map Classification. A total of 159 upregulated lipid species in IBS-D were detected within six lipid categories . A total of 475 downregulated lipids from seven lipid categories were observed: 78 fatty acids, 111 glycerolipids, 153 glycerophospholipids, 10 sphingolipids, 63 sterols, 58 prenol lipids, and 2 N-acyl amino acids ,D.p = 0.03). The Shannon index for IBS-D was slightly lower than that for HC, but the difference was not statistically significant . The patients/participants provided their written informed consent to participate in this study.HZ and YZ were performed the material preparation, data collection, analysis, and wrote the first draft of the manuscript. All authors commented on previous versions of the manuscript, contributed to the study conception, design, 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. 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.


To translate the Academic Goals Orientation Questionnaire (AGOQ) into Chinese and to determine the validity and reliability of the (AGOQ) in Chinese nursing students based on SEM and IRT multidimensional models.The participants were 654 nursing students with an age range of 17–26 years (mean age 21.61 ± 1.73 years). The psychometric properties of AGOQ were investigated based on a dual analytical perspective of structural equation modeling (SEM) and item response theory (IRT).The Cronbach’s α value of the questionnaire is 0.895. A four-factor model was obtained by exploratory factor analysis, which explained the variance of 71.892%. With confirmatory factor analysis, a new four-factors model was built and showed an acceptable goodness-of-fit, chi-square/degree of freedom (CMIN/DF) = 4.008, goodness of fit index (GFI) = 0.932, adjusted goodness of fit index (AGFI) = 0.905, comparative fit index (CFI) = 0.952, incremental fit index (IFI) = 0.952, Tucker Lewis index (TLI) = 0.941. In the analysis part of IRT, according to the comparison between Akek’s information criterion (AIC) and Bayesian information criterion (BIC), we choose the Graded Response Model (GRM) for analysis. The results show that the difficulty value is monotonically increasing, and the discrimination of all items is greater than 0.19, which shows that 16 items can be retained.This study tested the psychometric characteristics of AGOQ of nursing students in China. The results confirmed that the Chinese version of AGOQ has good psychometric characteristics and can be used to measure the academic goal orientation of nursing students in China.The online version contains supplementary material available at 10.1186/s12912-023-01630-0. The national standard for teaching quality of undergraduate majors in colleges and universities  requiredtask-oriented and self-oriented [Academic goals were defined as the content and direction of one’s motivation for academic success or failure , 3, whicoriented , 7. Howeoriented  studied oriented  and 2020oriented , and theFirst, students with Type I goals  focused on intrinsic stimuli and sought to absorb knowledge, acquired skills, and gained a true understanding of the problem . In shorBarkur et al.  examinedHowever, no Chinese studies on this topic were found during the literature search, possibly due to a lack of validated tools to measure students’ orientation toward academic goals. Academic goals orientation questionnaire (AGOQ) was first developed by a Norwegian scholar , and wasTherefore, this study aims to translate the Spanish version of the academic goals orientation questionnaire (AGOQ) into Chinese and evaluate the psychometric properties of the AGOQ in Chinese nursing students based on SEM and IRT multidimensional models.Cross-sectional design and multi-stage sampling design were adopted in this study. From March to June 2023, a survey was conducted among nursing students in medical schools in Jinzhou, Liaoning Province, China. The investigators of this study are mainly nursing graduate students who conducted this study. They received unified training on how to use standardized language and guidance  Ego self-frustration goal , (ii) Ego self-enhancement goal , (iii) Work avoidance goal items 3, 8, 12, 15), and (iv) learning or task goals . A five-point Likert scale was used to mark the answers that best matched the subjects’ current state . The reliability of the original scale with Cronbach’s alpha (α) > 0.8 in all dimensions was adequate [, 12, 15,There were various phases in the translation guide –20. FirsWe initially conducted a pre-survey and randomly selected 50 samples, and the following are the descriptive results of the pre-survey. The results of the pre-survey showed that the total score range of the scale is 16–64 (SD: 45.62 ± 11.10). The time to filled in the questionnaire is 3–6 min, with an average of 3.86 min. Supplementary material This study was completed between March and June 2023. The questionnaire included the Chinese version of AGOQ and socio-demographic information. This study adopted multi-stage sampling design. First, Jinzhou Medical University was randomly selected from 6 nursing colleges in Liaoning Province. Next, 50% of classes in each grade (ranging from one to three grade) were selected from the university , includiSPSS 25.0, AMOS 23.0, and R 4.3.0 were employed to analyze the statistics. Cronbach’s alpha (α) –24 was uExploratory factor analysis (the main component of Varimax rotation) , 26 was To measure model fit in CFA, eight indices were used: chi-square/degree of freedom (χ2/df), goodness of fit index (GFI), adjusted goodness of fit index (AGFI), incremental fit index (IFI), Tucker-Lewis index (TLI) , and comIn order to evaluate the AGOQ, IRT models were used. Graded Response Model (GRM) and Generalized Partial Credit Model (GPCM)  were exaTable Table Table χ2 = 6157.990; P < 0.001) [Table < 0.001) . The exp< 0.001) . Four faTable The results of confirmatory factor analysis (CFA) are shown in Table P < 0.001).In our study, the scores of the top (50%) and low (50%) groups were analyzed using a two-tailed independent samples t-test. As can be seen in Table In order to evaluate the AGOQ, IRT models were used. Graded Response Model (GRM) and Generalized Partial Credit Model (GPCM) were examined for improved model fit using AIC and BIC, whose values are lower suggesting a better model fit. The AIC and BIC values for GPCM in the current study were 27,259 and 27,617, whereas those for GRM were 27,145 and 27,504, respectively. The GRM was used as a result since it had a better model fit. According to Table The item characteristic curves and item information curves for the Chinese AGOQ are shown in Figs. The literature in nursing research links personal characteristics  and other factors  with academic burnout , droppinAs far as we know, this is the first study on academic goals orientation questionnaire (AGOQ) among nursing students in China based on structural equation modeling (SEM) and item response theory (IRT) model. The test results show that the Chinese version of AGOQ has good psychometric characteristics and is an effective and reliable tool. These results are consistent with the original version developed by Skaalvik  and the Previously, Elliot  developeP < 0.001). In addition, each item of AGOQ has higher load value and commonality coefficient. The results also indicated that there were strong factor loadings and explained variance in the structural equation modeling, consistent with the EFA results.In the exploratory factor analysis (EFA) model, this study extracted four factors which are the same as the original scale. The four factors explained 71.892% of the total variance, 20.256%, 19.788%, 17.099% and 14.748%, respectively. The measured values of the model fit well (chi-square/degree of freedom (CMIN/DF) = 4.008, comparative fit index (CFI) = 0.952, incremental fit index (IFI) = 0.952, Tucker Lewis index (TLI) = 0.941). The results showed that the model has strong factor load and explanatory difference. The results of confirmatory factor analysis (CFA) confirmed that the Chinese version of AGOQ had a fitting index. There was significant difference in discriminant validity between the high group and the low group


The aim was to explore the inter-reliability of a newly developed US scanning protocol  for the assessment of different aspects of sarcopenia-related muscle involvement, including muscle mass, muscle quality and muscle stiffness [using point shear-wave elastography (SWE)], in patients with rheumatic and musculoskeletal diseases (RMDs).Quadriceps muscle mass (i.e. muscle thickness), muscle quality  and point SWE measurements were obtained by two rheumatologists  in consecutive RMD patients without previous/current myositis or neuromuscular disorders.Inter-reliability was assessed using the intraclass correlation coefficient (ICC) for continuous variables and Cohen’s kappa (κ) for categorical variables.2], 10 with PsA, 7 RA, 5 AS, 5 PMR, 4 SLE, 4 gout, 4 OA, 3 FM and 3 SSc. The grade of inter-rater reliability was excellent for muscle mass [ICC = 0.969 (0.953 < ICC < 0.979)]. Regarding muscle echogenicity, the agreement was substantial/almost perfect using the visual semi-quantitative scale  and excellent using ImageJ analysis [ICC = 0.916 (0.876 < ICC < 0.944)]. Finally, a good agreement was obtained for point SWE measurements [ICC = 0.76 (0.712 < ICC < 0.8)].A total of 45 RMD patients were enrolled [mean age 54.5 (16.0) years, male-to-female ratio 1:1.5, mean BMI 24.6 (4.6) kg/mMultimodal US is a novel and reliable tool for the evaluation of different aspects of muscle involvement  in RMD patients. Key messagesMultimodal US enables the simultaneous assessment of different aspects of muscle involvement, including muscle mass, muscle quality and muscle stiffness.The study showed a good to excellent inter-reliability of multimodal US in RMD patients.Multimodal US is a promising and reliable tool for the detection of muscle involvement in RMD patients.Sarcopenia is characterized by progressive and generalized loss of skeletal muscle mass and strength. It is associated with physical disability, poor quality of life and increased mortality . SarcopeWhen secondary to systemic inflammatory diseases, such as RA, sarcopenia might occur at a younger age compared with the general population, as the consequence of pain and decreased mobility, increased production of pro-inflammatory cytokines, and muscle tissue degradation secondary to cellular metabolic alterations , 4.MRI, CT and DXA are regarded as reference imaging tests for the detection of sarcopenia , 6. TheiUS can depict muscle anatomical details and both quantitative (i.e. decreased muscle mass) and qualitative  muscle changes . FurtherPrevious studies have demonstrated the potential value of US in the detection of sarcopenia; most of these studies were carried out in geriatric populations, and the applications of this imaging tool in patients with rheumatic and musculoskeletal diseases (RMDs) have scarcely been investigated .In a recent study, our research group developed a new US scanning protocol for the assessment of multiple aspects of sarcopenia-related muscle involvement, including muscle mass, muscle quality and muscle stiffness  . In thisBesides the promising clinical implications this new US protocol (i.e. early detection of sarcopenia-related muscle involvement in RMD patients), its inter-reliability (defined as the degree of agreement between different operators) remains to be determined. Thus, the aim of this study was to evaluate the inter-reliability of multimodal US for the assessment of muscle involvement  in RMD patients.Consecutive patients attending the outpatient clinic of the Rheumatology Unit of ‘Carlo Urbani’ Hospital, in Jesi  were included. Enrolment of patients started in March 2021 and ended in April 2021. Exclusion criteria were age <18 years, history of myositis or neurodegenerative disorders, and intense physical activity in the week preceding the study enrolment. This study was approved by the local ethics committee, Comitato Etico Regione Marche (CERM no. 155/2021). All the patients gave their informed consent.Two rheumatologists  performed a bilateral US assessment of the quadriceps muscle, blinded to each other’s evaluations.As previously reported , the US Rectus femoris (RF) and vastus intermedius (VI) muscle thicknesses were measured, in addition to their sum (quadriceps muscle thickness), adopting the anterior superior iliac spine (ASIS) technique . This teEchogenicity of the RF and VI muscles was assessed using a modified version of the Heckmatt scale . This moMuscle echogenicity was also determined using ImageJ (v.1.53e) by both US operators. This software, which is a public-domain Java-based image analysis program, measures the greyscale intensity in a region of interest using a histogram function . The RF Measurements of SWE were obtained with the probe oriented longitudinally at four distinct muscle quadrants of the RF muscle  and repeated three times for each muscle area evaluated. Therefore, a total of 24 SWE acquisitions  was obtained in the included patients. The quadrants were identified using the midpoint between the ASIS and upper pole of the patella as the starting point. From this midpoint, the probe was translated 1 cm medial to the central aponeurosis , then 1 cm laterally , then 2 cm proximal to medial  and, finally, 2 cm proximal to lateral . There was no predefined order to the sequence for obtaining SWE, but distal measurements  always had to be obtained before proximal measurements . The median of the three measurements for each area was recorded as SWE velocity in units of metres per second, with the corresponding interquartile range. The regions of interest for SWE measurements and depth were placed between the superficial fascia of the RF muscle and the interface with the VI muscle.Inter-operator reliability for continuous variables  was assessed using the intraclass correlation coefficient (ICC), with the 95% confidence interval. For categorical, ordinal variables , weighted Cohen’s kappa (κ) was calculated. Finally, the statistical association between echogenicity measures  was probed using Spearman’s correlation coefficient. All estimations were performed using R and RStudio (R v.4.1.2).The κ coefficients were interpreted according to Landis and Koch  .A total of 45 RMD patients were enrolled consecutively  for the RF muscle, 15.4 (5.2) mm for the VI muscle, 33.3 (8.2) mm for the quadriceps muscle (RF + VI) . According to the operator with more years of experience in the use of US, muscle echogenicity evaluated using the semi-quantitative visual scale had the following distribution: 16% rated as 0 ; 37% rated as 1 (mild); 31% rated as 2 (moderate); and 16% rated as 3 (severe). Muscle echogenicity evaluated using ImageJ analysis was equal to 93.2 (13.8) [mean (s.d. )]. Finally, muscle stiffness, as measured by SWE values for each quadrant, was 1.42 (0.24) m/s , 1.53 (0.27) m/s , 1.45 (0.28) m/s  and 1.53 (0.26) m/s .The muscle thickness was 17.9 (4.2) mm [mean (P < 0.001).The visual semi-quantitative assessment and the ImageJ-derived echogenicity measures for muscle echogenicity showed a significant positive association ]. Regarding muscle echogenicity, the agreement was substantial/almost perfect using the visual semi-quantitative scale  and excellent using ImageJ [ICC = 0.916 (0.876 < ICC < 0.944)]. Finally, a good agreement was found for point SWE [ICC = 0.76 (0.712 < ICC < 0.8)].The results of this live (i.e. patient-based) study showed a good to excellent reliability of a recently developed US scanning protocol for the assessment of muscle involvement in RMD patients. This newly developed US scanning protocol enables a combined assessment of different elements of muscle involvement, such as muscle mass, muscle quality and muscle stiffness. All these single elements could potentially be implicated in the pathogenesis of sarcopenia. Therefore, their simultaneous assessment appears to be relevant in patients with RMDs, in which different pathogenetic mechanisms leading to sarcopenia have been hypothesized, in comparison to elderly patients with primary (i.e. age-related) sarcopenia . InteresThere is a vast literature supporting the high reliability of US muscle mass assessment (i.e. muscle thickness measurement) . The ASIvs moderate, respectively), which is arguably more representative of real life if compared with a remote evaluation of static images and clips.In our previous study , we propFinally, SWE has a promising role in the assessment of muscle elasticity, and therefore muscle physiological and biomechanical status, in RMD patients. Methodological issues are regarded as the main limitations to the clinical applicability of SWE . AnisotrAs described in the Methods, point SWE measurements were obtained following a standardized and rigorous protocol, which arguably explains the good reliability results obtained for this assessment, although these reliability results were lower than those obtained for the more standardized (and arguably less subjected to variations) assessment of muscle thickness (i.e. good for SWE and excellent for muscle mass measurement).In a previous study, our newly developed multimodal US has emerged as a promising screening tool for the detection of sarcopenia-related muscle involvement in patients with SLE and, potentially, in RMD patients . FurtherOur study has some limitations. First, the two operators who performed the inter-reliability exercise were also those who developed the multimodal US. The external inter-reliability of this newly developed scanning protocol has yet to be investigated. Second, intra-reliability assessment was not performed. This was a real-world study, which included patients attending the outpatient clinic of our Rheumatology unit, and it was not possible to perform a repeated assessment on the same day (or on different days).Our recently developed multimodal US can reliably assess different aspects of muscle involvement  in RMD patients.


The objective of this study was to compare the graft outcome and postoperative infection of with and without the use of antibiotic ointment following myringoplasty for the treatment of chronic perforations.Randomized controlled trial.135 chronic perforations were prospectively randomized to use of antibiotic ointment group  or no use of antibiotic ointment group  following myringoplasty. The graft outcomes and postoperative infection were compared among two groups at 6 months.P = 0.312).The graft success rates were 92.6% in the UAO group and 91.0% in the NAO group, the difference was not significant (P = 0.979). In the UAO group, 3 patients with purulence ear discharge resulted in a residual perforation although they received ofloxacin ear drops and intravenous antibiotic therapy treatment. In the NAO group, 6 patients with purulence ear discharge resulted in a residual perforation, only one, with mild purulence discharge was successfully treated and closed. In addition, no significant between-group differences were observed pre- (P = 0.746) or post- (P = 0.521) operative air bone gap (ABG) values or mean ABG gain (P = 0.745). However, granular myringitis with minimal moistness but without infection has been noted in 3 (4.4%) patients in the UAO group and in 5 (7.5%) in the NAO group, the difference was not significant (P = 0.699).At postoperative 6 months, the graft infection rate was 4.4% in the UAO group and 10.4% in the NAO group, the difference was not significant (Use and non-use of antibiotic ointments for lateral packing of graft are both comparable methods following myringoplasty for postoperative infection and graft outcomes. Myringoplasty has been successfully used to repair vairous perforation. Packing of the external auditory canal (EAC) after surgery is an established practice in most hospitals, this is believed to protect the operated site, hold the graft in situ , 2. The This prospective Randomized Controlled Trial (RCT) was reviewed and approved by the Medical Ethical Committee of Yiwu central hospital. Informed consent was obtained from all participants.Study subjects were recruited from consecutive adult patients diagnosed with chronic perforation who visited the Department of Otorhinolaryngology, Head and Neck Surgery. The inclusion criteria was included: no marginal small-or medium size perforation with no involving the malleus umbo; dry ear at least 3 months prior to surgery based on patient history, good pneumatization of the middle ear and mastoid region, as shown by high-resolution computed tomography (HRCT). Prior to enrollment, HRCT was performed, and audiograms were recorded in all patients. Exclusion criteria were active inflammation during the 3 months prior to surgery, revision surgery, cholesteatoma, and requiring ossiculoplasty. The perforation size was categorised as small (< 1/8 of the TM area), medium (1/8–1/4 of the TM area) , 7. The Audiometric data were obtained preoperatively and 6 months postoperatively. Pure-tone averages (PTAs) were calculated for both air conduction (AC) and bone conduction (BC) by averaging the thresholds at 500, 1000, 2000, and 3000 Hz; in most cases, a threshold of 4000 Hz was used to interpolate the threshold of 3000 Hz per the standards of the Hearing Committee of the American Academy of Otolaryngology-Head and Neck Surgery. Pre- and postoperative air bone gap (ABG) was calculated by subtracting the AC PTA from the BC PTA. ABG closure was calculated as preoperative ABG minus postoperative ABG ) or percentages (%). The chi-squared test was used to compare categorical data. The Wilcoxon and Mann–Whitney U-tests were employed to compare non-parametric variables and the independent and paired samples t-tests were used to compare parametric variables. A A total of 135 chronic perforations were included. Of the 135 ears, 68 perforations were in the UAO group and 67 perforations in the NAO group  .Non-purulent exudate of varying degrees was seen in both groups at postoperative 1–4 weeks, except for the perforations with graft infection. The graft success rates were 92.6% (63/68) in the UAO group and 91.0% (61/67) in the NAO group, the difference was not significant (P = 0.746) or post- (P = 0.521) operative ABG values or mean ABG gain (P = 0.745). Also, no significant between-group differences were observed pre- (P = 0.617) or post- (P = 0.639) operative mean BC PTA.The postoperative ABG showed significant improvement compared to pre-operation in both groups, while no significant difference was observed in any group with regard to pre- and post-operation average BC PTA Table . In addiP = 0.699).During followup period, no complications were observed in both groups, including deteriorative sensorineural hearing loss, altered taste, vertigo, or tinnitus. Also, no graft lateralization, significant blunting or medialization were found in any group. All the patients didn’t develop postoperative otitis media effusion and graft retraction. However, granular myringitis with minimal moistness but without infection has been noted in 3 (4.4%) patients in the UAO group and in 5 (7.5%) in the NAO group, the difference was not significant . Thus, there was no documented evidence of the use of antibiotic ointment affecting graft infection following myringoplasty. This result was similar to previous findings. Hirschmann et al. [P = 0.490) and putting ointment on a surgical wound does not benefit the patient. Kalmeijer et al. [Some scholars recommended that topical antibiotic ointments are effective to use on open wounds in preventing wound infections . Howevern et al.  found thn et al.  comparedr et al.  performeIn the field of otologists, Cunningham et al.  applied In this study, Erythromycin ointment was choosed to pack lateral surface of the graft. Most of literatures reported that Staphylococcus aureus and Pseudomonas aeruginosa were the commonest bacteria for chronic suppurative otitis media (CSOM) –24, howeAlthough some scholars reported that the graft success rates following myringoplasty was not affected by the presence of preoperative otorrhea , 25, posApparently, mean ABG gain was not significantly different regardless of the use of antibiotic ointment or not. Also, similar with previous study , 10, no These findings may challenge commonly held surgical beliefs about the need for lateral packing following myringoplasty. The advantages of this study is that no patient was lost to follow-up from each group, thus supporting the results. In addition, the graft change may be clearly observed at different followup points by endoscope because of the absence of EAC packing of biodegradable synthetic polyurethane foam. However, the limitations of this study was small sample size, possibility of bias due to lack of blinding, and short-term followup. In addition, bacterial culture and antimicrobial susceptibility testing were not performed for the patients with purulence discharge in both groups.Use and non-use of antibiotic ointments for lateral packing of graft are both comparable methods following tympanoplasty for postoperative infection and graft outcomes.


Women with Turner syndrome (TS)  have an increased prevalence of conditions such as diabetes mellitus, obesity, hypothyroidism, autoimmunity, hypertension, and congenital cardiovascular anomalies (CCA). Whilst the risk of developing these co-morbidities may be partly related to haploinsufficiency of key genes on the X chromosome, other mechanisms may be involved. Improving our understanding of underlying processes is important to develop personalized approaches to management.TIMP3 variants with CCA can be replicated.We investigated whether: 1) global genetic variability differs in women with TS, which might contribute to co-morbidities; 2) common variants in X genes - on the background of haploinsufficiency - are associated with phenotype (a “two-hit” hypothesis); 3) the previously reported association of autosomal TIMP3 variance was investigated in relation to CCA.Whole exome sequencing was undertaken in leukocyte DNA from 134 adult women with TS and compared to 46,XX controls (n=23), 46,XX women with primary ovarian insufficiency (n=101), and 46,XY controls (n=11). 1) Variability in autosomal and X chromosome genes was analyzed for all individuals; 2) the relation between common X chromosome variants and the long-term phenotypes listed above was investigated in a subgroup of women with monosomy X; 3) TIMP3 22:32857305:C-T and CCA was found .Standard filtering identified 6,457,085 autosomal variants and 126,335 X chromosome variants for the entire cohort, whereas a somatic variant pipeline identified 16,223 autosomal and 477 X chromosome changes. 1) Overall exome variability of autosomal genes was similar in women with TS and control/comparison groups, whereas X chromosome variants were proportionate to the complement of X chromosome material; 2) when adjusted for multiple comparisons, no X chromosome gene/variants were strongly enriched in monosomy X women with key phenotypes compared to monosomy X women without these conditions, although several variants of interest emerged; 3) an association between TIMP3 variance and congenital cardiac anomalies was replicated.Women with TS do not have an excess of genetic variability in exome analysis. No obvious X-chromosome variants driving phenotype were found, but several possible genes/variants of interest emerged. A reported association between autosomal  The agemphedema . Others mphedema . Prompt mphedema , 9.In adulthood, women with TS also have a higher prevalence of common conditions such as diabetes mellitus (DM), autoimmunity, hypothyroidism, hypertension, and cardiovascular disease , 8, 10. The association between karyotype and phenotype in women with TS is still unclear. Karyotypes found in TS include aneuploidies , as well as structural X chromosome variants  or 45,X/46X,i(Xq) mosaicism [10%], 45,X/46,X,r(X) ring mosaicism [7%], or rarer Xp and Xq deletions) . Several5%, 45,X/-10%, 45,The mechanism (or mechanisms) by which aneuploidy or structural variance of the X chromosome gives rise to the broad range of phenotypes in TS is still under investigation , 19–22.left illustration). PAR1 genes are typically haploinsufficient in all women with non-mosaic TS, irrespective of karyotype , homologous regions present on the short arm (PAR1) and long arm PAR2) of both sex chromosomes (X and Y)  of both , 24 , translation , splicing  and DNA methylation/chromatin modification , with ZFY having a potential key regulatory role in this regard  in a “two-hit” mechanism .1) Whether disruption of an X chromosome gene with a proofreading or DNA repair function has a wider “ripple” effect on the genome, resulting in increased genetic variability that might contribute to excess morbidity and other features (right illustration). Having a single copy (haploinsufficiency) of a gene in this region may already infer a risk for a given phenotype. We hypothesize that in this scenario, common population variants in the remaining expressed allele could have an unexpectedly strong influence on phenotype, as this is a unique biological situation where they are present in a hemizygous  state. Thus, the functional influence of common variants could be “exposed” and have quite a marked effect on the risk of developing conditions such as DM and autoimmunity (“X chromosome two-hit hypothesis”).2) Can common hemizygous variants in X chromosome genes  contribute to the risk of associated features in TS  are associated with an increased risk of congenital cardiac anomalies (“autosome two-hit hypothesis”), as has been reported previously (3) If variants in the autosomal gene eviously , 36.In order to investigate these hypotheses further, we undertook a large-scale genetic analysis in 134 women with monosomy X and associated karyotypes and investigated genetic variability in relation to control groups and phenotypic features.22.12.1.1The study was conducted as part of the Reproductive Life Course Project ) at University College London Hospitals (UCLH), London, UK. A total of 134 women were recruited from the UCLH Turner Syndrome clinic (2015-2019) overseen by specialist multi-disciplinary professionals. All women provided written, informed consent to take part .The original diagnosis was made by G-banded karyotype analysis undertaken by routine clinical cytogenetic services. A mosaic screen of at least 30 cultured lymphocytes was typically done. Following recruitment to this study, single nucleotide polymorphism (SNP) array analysis was undertaken on a recent leukocyte-derived DNA sample (see below). Women were excluded if they had evidence of 46,XX or 46,XY mosaicism, or if a Y fragment was identified on original karyotype or on recent SNP array analysis. Women with complex structural variance or complex aneuploidies with a 46,XX or 47,XXX cell line were also excluded.Within this cohort, three women who were originally reported to have a non-mosaic 46,X,i(Xq) karyotype were subsequently found to have a low-level mosaic  line on SNP analysis ). Three other women had karyotypes that were discordant with the original results, but in these cases historical records were limited. No women had a significant Y line or 46,XX line present. In all situations the most recent SNP array karyotype was used for grouping in this study Table 1.Key clinical parameters that were chosen for further analysis in relation to genetic variability were DM/impaired glucose tolerance (IGT), obesity, autoimmune disease, hypothyroidism, hypertension, congenital cardiovascular anomaly, and hearing loss. These conditions were defined as:1) Diabetes mellitus: A fasting plasma glucose of ≥7.0 mmol/l (126 mg/dL), or ≥11.1 mmol/l (200 mg/dL) after 120 minutes on a standard oral glucose tolerance test . Impaired glucose tolerance: A plasma glucose of between 7.8 mmol/l (140 mg/dL) and 11.1 mmol/l (200 mg/dL) after 120 minutes on a standard oral glucose tolerance test . For the purposes of analysis, we combined individuals with diabetes and individuals who had IGT under the label “Diabetes”.2.2) Obesity: A body mass index (BMI) greater than 30 kg/m3) Autoimmunity: A diagnosis of autoimmune disorders such as celiac disease, inflammatory bowel disease, or antibody positive hypothyroidism.4) Hypothyroidism: An elevated TSH and long-term treatment with thyroxine replacement. The size of this subgroup allowed for this to be analyzed as a separate entity and may have included women with autoimmune hypothyroidism who were no longer auto-antibody positive.5) Hypertension: A persistent elevation in blood pressure (140/90 mmHg or higher) and treated with long-term antihypertensive therapy.6) Congenital Cardiovascular Anomaly (CCA): The presence of a bicuspid aortic valve or coarctation of the aorta or any form of cardiac surgery in childhood. Unfortunately, serial aortic root dimension data in adulthood were not available for analysis.7) Hearing loss: The use or recommended use of a hearing aid.2.1.2For comparisons of global genetic (exome) variability, data from 101 women with POI were obtained from the Reproductive Life Course Study at University College London Hospitals, London. Those with a known cause of ovarian dysfunction  were excluded . Women r2.1.3Control DNA samples  were obtained from Human Random Control DNA Panels .2.2Total DNA was extracted and purified from whole blood using a QIAamp Blood Maxi Kit or QIAamp Blood Midi Kit , following the manufacturer’s protocol. Samples were submitted for SNP array and exome sequencing through UCL Genomics.2.3SNP array analysis was undertaken using Illumina Global Screening Arrays (v3.0) containing 654,027 markers, following the Infinium HTS Assay Reference Guide # 15045738 v04) . Raw data files were analyzed in Illumina Genome Studio version 2.0 and X chromosome mosaicism calculations were made using methods described by Conlin et al.  and Nonacus protocol (Protocol Guide v1.2.2) with minor modifications. In brief, 200ng of genomic DNA was used for exome pre-capture library preparation. Library preparations were carried out on a Hamilton StarLet robotic platform  and library qualitative checks were undertaken using Tapestation 4200 . Sample libraries were sequenced on a NovaSeq6000 Platform using an S4 flow cell (Illumina).2.4.2FASTQ files were generated and processed through the bioinformatics pipeline shown in Generated VCF files were uploaded to the Qiagen Clinical Insight (QCI) Interpret web-based platform for variant annotation and classification. All exome variants for all samples were exported from QCI as CSV files, these files were then merged and underwent two parallel approaches for standard variant and somatic mosaic variant filtering Figure 22.4.3post-hoc power analysis based on final group size and ratios of those with and without a condition (https://www.omim.org). Gene expression data for genes of interest were obtained using the consensus summary in the Human Protein Atlas (https://www.proteinatlas.org).To investigate whether genetic variants in X chromosome genes  are associated with phenotype, X chromosome variants from the standard filtering pipeline Figure 2Following the initial analysis of X chromosome variants linked to phenotype in the 45,X monosomy subgroup, a separate analysis of PAR gene variance was undertaken in a wider cohort of women with additional TS-associated karyotypes using a similar design, as PAR1 genes are predicted to be haploinsufficient in all these women.Analysis of “hearing loss” was also undertaken using the same pipelines (14 with versus 33 without), but these data are presented separately as the groups are smaller and the end points potentially less reliable.2.5TIMP3 (chromosome 22) variants and CCA, exome sequencing data for variants in this gene were analyzed in relation to cardiac status in 95 women where phenotypic data were available. The minor allele frequency (MAF) of common variants detected was calculated and compared between groups (CCA [n=22] versus non-CCA [n=73]) using Fisher’s Exact testing. MAF was also compared to data obtained from gnomAD (v3.1.2) as well as previous reports of TIMP3 variants in TS cardiac cohorts  using the Wilcoxon matched-pairs signed rank test.Where data were available, statistical analysis between the original and new percentage mosaicism values was undertaken in GraphPad Prism  test in GraphPad Prism. For X chromosome variant filtering, and gene and variant level counts, Fisher and Bonferroni adjustment tests were undertaken in R using the tidyverse packages , 41. Bon2.6.3TIMP3 related to CCA was performed using Fisher’s Exact tests in GraphPad Prism.Statistical analysis for potential enrichments of variants in 33.1An overview of the study cohort and sub-groups is shown in 3.2center illustration), total autosomal and X chromosome variants were compared between cohorts as well as the potential translational impact of any changes.To investigate whether disruption or changes in the X chromosome in TS affect genetic (exome) variation in general , we initially focused subsequent analysis only on women with monosomy X  who had detailed phenotypic data available for DM, obesity, autoimmunity, hypothyroidism, hypertension and cardiac surgery for congenital heart defects. Only women with monosomy X were included in the initial analysis as they have just a single copy of the X chromosome, avoiding any issues with multi-allelic expressed genes and allowing us to focus on hemizygous variants that could act as important drivers of phenotype especially on the background of haploinsufficiency of PAR genes and of genes that escape Xi. As we hypothesized that common population variants could have a major effect in this biological context, cohorts of 45,X women with and without a given phenotype  were directly compared for enrichment of X chromosome genetic variants that could act as “risk” or “protective” alleles in this situation.To address whether common hemizygous variants in X chromosome genes contribute to associated features in TS  are shown in MIR4769 and F8, whereas “protective” genes were DOCK11 and STEEP1. In the obesity group, LANCL3, a hypothalamic expressed gene associated with carbohydrate metabolism , splice analysis or Combined Annotation Dependent Depletion (CADD) scores (Despite the lack of significance following multiple corrections (for more than 600 genes), further review of the expression, biological role and clinical associations of these genes was undertaken Table 2.tabolism  and RPL3yroidism , potentiAKAP17A, linked to autoimmunity and showed non-adjusted significance in the variant level analysis   in lue 1.0) 4. Heari3.4TIMP3 in relation to cardiac anomalies  revealed a significant enrichment of the 22:32857305:C-T (rs11547635) variant in the CCA group  (https://spliceailookup.broadinstitute.org/). A proposed combined effect of this TIMP3 variant with TIMP1 copy number (1.0) for cardiac risk (TIMP1 copy number) from the cohort. Notably, there was a statistically higher proportion of women with monosomy X in the CCA group, and lower proportion of those with ring chromosomes  Table 3. rs115476 variant CRELD1  has been reported to be associated with CCA in women with TS  leading to these comorbidities is still not clear. Often this is assumed to be haploinsufficiency of PAR genes on the X chromosome, but other subtle mechanisms related to X gene dosage or autosomal/X-chromosome “ripple effects” may be important . By usinBefore engaging in in-depth genomic analysis, our first goal was to assess the karyotypes of this cohort of women using a SNP array approach, rather than the traditional G-banded karyotyping and 30 cell mosaic screen. Using this platform, a current karyotype was determined for all participants. Three women  originally reported to have non-mosaic isochromosomes ) were found to have a low-level mosaic 45,X line present on array ). Three additional women had other changes in karyotype, but original records were limited, or small numbers of cells screened. Overall, SNP array proved to be a useful approach to assessing karyotype for this cohort and there was concordance with original recorded karyotype in more than 95% individuals.In the SNP array dataset, mosaicism levels for ring and isochromosomes were calculated using the approach reported by Conlin et al. . As expeHaving defined karyotypes in the cohorts, targeted exome sequencing was used to test the hypothesis of whether changes in the X chromosome with TS are associated with differences in global genomic variability either across the autosomes or in the X chromosome itself. It has been hypothesized that haploinsufficiency of key X chromosome genes could have “ripple effects” affecting transcription, translation, splicing or methylation/chromatin across the genome , 47, 48.VDR, ESR1); response to growth hormone treatment (GHRd3)) , or conversely whether changes could be found more often in women without a given condition compared to those with it .LANCL3. This gene has been linked to carbohydrate metabolism/high fat diet induced obesity in rats, is expressed in the hypothalamus and has been shown to escape X inactivation in TS, so further investigation may be warranted (AKAP17A (X:1601004:C-G) associated with autoimmunity. This gene has been implicated in inflammatory bowel disease  and statistical differences did not withstand multiple comparison testing. Furthermore, biological correlations of proposed gene function with phenotype were not obvious, except in the case of obesity and arranted , 42. The disease  but thisTIMP3 and congenital cardiovascular anomalies in two cohorts of women with TS, we undertook a specific analysis of TIMP3 and CCA in our study group. TIMP3 encodes Tissue Inhibitor of Metalloprotease 3, an extracellular matrix protein involved in angiogenesis and cardiac remodeling (TIMP3 gene is located on chromosome 22. It is hypothesized that loss of TIMP1 (on the X chromosome) coupled with variations in TIMP3 can predispose to CCA in women with TS, in an autosomal “two-hit” hypothesis (TIMP3 variant is relatively low (CCA 0.14 versus non-CCA 0.03) and effect size small (0.11). Our X chromosome variant analysis was far more stringent, focusing on an effect size cut off at +/- 0.35 and adjusting for multiple comparisons for all X genes. Given the TIMP3 data, several of the variants we detected in the X chromosome may be biologically significant. This could be addressed in the future in larger cohorts, combining our datasets with additional cohorts.Given a previously reported association between the 22:32857305:C-T rs11547635) variant in modeling , 51. The variant pothesis . Very repothesis . Notablypothesis Table 3.pothesis . This ficommon population variants in X chromosome genes are exposed and contribute to phenotype. However, the power to detect statistical differences is dependent on sample number, and potentially important changes with a smaller effect size may have been missed. As discussed above, the potential influence of TIMP3 on congenital cardiac phenotypes was replicated, even for a variant of low allele frequency and small effect size. Fourth, some studies have suggested that tissue mosaicism for a covert 46,XX or 46,XY cell line may influence phenotype  and metabolic effects Type 2) in diabetes mellitus), or be inter-dependent . In order to improve phenotype accuracy, gold standard approaches and detailed re-evaluations were undertaken, such as oral glucose tolerance testing to capture women with undiagnosed diabetes as well as those on established treatment. Only women with robust phenotypic data were included for analysis. Secondly, variant numbers can be influenced by sequencing quality  and batch effects. We compensated for this by processing all samples at the same time and using robotic approaches to undertake library preparations. Third, the numbers of individuals in the phenotype groups for X chromosome variant analysis were relatively small and the power of burden testing when adjusted for multiple comparisons would have been stronger if more individuals had been included. Our hypothesis was that  in diabeDespite these limitations, this is a major hypothesis-based study using SNP array and exome sequencing in one of the largest populations of women with TS studied at a detailed genetic level to date. This work highlights how rapidly advancing technologies can increasingly be applied at scale to address key questions in well-characterized clinical cohorts, with the aim of developing more specific or personalized approaches to treatment for associated conditions in the future.https://doi.org/10.17605/OSF.IO/AJBEP, Open Science Framework (The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: ramework .The studies involving humans were approved by NRES Committee London-Chelsea (16/LO/0682). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.JS, GC and JA conceptualized the study. JS, AP-C, SM-B, MI, ID, FB, TB, GK, GC and JA undertook data curation. MI and JS undertook formal data analysis. GC and JA were involved in funding acquisition. AP-C, GC and JA undertook investigation, diagnosis, and management. GC and JA oversaw project administration and supervision. MI and ID undertook validation. JS and FB were responsible for data visualization. JS, FB and JA wrote the original draft with input from GC. All authors were involved in reviewing and editing the final manuscript. GC and JA had full access to all data in the study and had final responsibility for the decision to submit for publication. All authors contributed to the article and approved the submitted version.


At baseline, local activity, as quantified by fractional amplitude of low-frequency fluctuations, differentiated between normal and abnormal CSF groups in the NbM but not EC. Further, NbM activity linearly decreased as a function of CSF ratio, resembling the disease status. Finally, NbM activity predicted the annual percentage signal change in EC, but not the reverse, independent from CSF ratio. Our findings give novel insights into the pathogenesis of Alzheimer’s disease by showing that local activity in NbM is affected by proteinopathology and predicts functional degeneration within the EC.Recent models of Alzheimer’s disease suggest the nucleus basalis of Meynert (NbM) as an early origin of structural degeneration followed by the entorhinal cortex (EC). However, the functional properties of NbM and EC regarding amyloid-β and hyperphosphorylated tau remain unclear. We analysed resting-state functional fMRI data with CSF assays from the Alzheimer’s Disease Neuroimaging Initiative  differentiates between normal and abnormal CSF and decreases linearly with CSF ratio. NbM activity predicts signal changes in the entorhinal cortex, but not vice versa, independent of CSF ratio, offering novel insights into Alzheimer’s disease pathogenesis.Mieling   To this end, we used data from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) and performed a longitudinal region of interest (ROI) analysis over 2 years, focusing on regional and interregional resting-state functional MRI (rsfMRI) properties. In detail, we analysed (i) the fractional amplitude of low-frequency fluctuations (fALFF) to quantify spontaneous neuronal activity,10-12 (ii) regional homogeneity (ReHo) reflecting the synchronicity of neural activity between a voxel and its neighbouring voxels13 and finally (iii) the functional connectivity between NbM and EC. While all three measures may help to gain new insights into Alzheimer’s disease progression, we initially focused on fALFF given its established role14-16 and report ReHo and functional connectivity analyses in the Since functional brain changes in Alzheimer’s disease often precede structural degeneration,In the first step, baseline signals and longitudinal functional changes were compared based on harmonized CSF assays of Aβ and pTau in NbM and EC. Subsequently, we investigated functional changes in disease progression using the CSF markers. Finally, we tested the competing models NbM→EC versus EC→NbM on a functional level. Our main hypothesis was that functional signals in the NbM predict functional changes in EC, which would provide further evidence supporting the pathological staging model from NbM to EC. From a more general perspective, we aimed to provide new insights into the underlying functional properties of Alzheimer’s disease, which may contribute to further developing markers and treatment strategies.Data used in the preparation of this article were obtained from the ADNI database (adni.loni.usc.edu). ADNI was launched in 2003 as a public–private partnership, led by Principal Investigator Michael W. Weiner, MD, to test whether serial MRI, PET, other biological markers and clinical and neuropsychological assessment can be combined to characterize the progression of mild cognitive impairment (MCI) and early Alzheimer’s disease.https://ida.loni.usc.edu). Specifically, we only selected data from participants with CSF biomarkers and two rsfMRI scans acquired with a delay of 2 years with the same MR scanner and head coil to ensure within-subject comparability.Since rsfMRI was not acquired in all ADNI cohorts, here, data were combined from ADNI-GO, ADNI-2 (ADNI-GO/2) and ADNI 3, downloaded from the Image and Data Archive (IDA) platform run by the Laboratory of Neuro Imaging (LONI) . Further, we only included MRI data with excellent, good or fair quality. For further information on image acquisition, see the http://adni.loni.usc.edu.Participants were scanned at multiple sites equipped with 3-Tesla MRI scanners according to unified ADNI monitoring protocols.http://rfmri.org/dpabi) toolbox version 5 (release 5.2_210501), which is based on the Statistical Parametric Mapping toolbox  for MATLAB®. It started with the removal of the first 10 volumes and subsequently included the following steps (i) slice time correction; (ii) spatial realignment; (iii) T1 co-registration to the mean functional image; (iv) CSF, grey and white matter tissue segmentation and spatial normalization using diffeomorphic anatomical registration using exponential lie algebra (DARTEL)18 for T1 images; (v) regression of nuisance variables; and (vi) normalization to the Montreal Neurological Institute (MNI) space and resampling to an isotropic voxel size of 3 mm of the functional images using the parameters estimated by DARTEL  by overlaying a ROI mask in standardized space; especially, the EC represents a region that might often be affected by artefacts.21 To better understand how both relate to functional degeneration in NbM and EC, we followed previous studies2 and used ADNI’s CSF samples, produced with a fully automated Elecsys® protocol of Aβ and pTau from the first measurement (t1). For each participant, we extracted Aβ 1–42 and pTau181 values. Since the protocols by Elecsys® are still under development, the results are restricted to a specific technical limit (>1700 pg/mL). Higher values were provided by extrapolation of the calibration curve for research purposes only but not diagnostics. Further information on CSF draws and analyses can be found at http://adni.loni.usc.edu.Alzheimer’s disease neuropathology includes the accumulation of Aβ resulting in plaques and pTau leading to neurofibrillary tangles.23 Based on these findings, the standardized and cross-validated cut-off of 0.028 was used to divide the participants into an abnormal (pTau/Aβ ≥ 0.028) and a normal (pTau/Aβ < 0.028) CSF group.23 Importantly, no participant classified with Alzheimer’s disease had a normal CSF ratio, but a few (n = 10) participants classified with MCI did, which indicates an unclear aetiology. Nevertheless, we included them based on biological instead of a syndromal grouping.24Here, we analysed both proteins by using a previously established ratio of pTau/Aβ, which is known to highly concord with PET measures and clinical diagnoses.t1 scores are used, including validated memory (MEM) and executive function (EF), based on a confirmatory factor analysis.26 Memory scores include the Alzheimer’s Disease Assessment Scale, Logical Memory test, Mini-Mental State Examination (MMSE) and Rey Auditory Verbal Learning Test (RAVLT). EF scores are based on the Category Fluency, Digit Span Backwards, Digit Symbol Substitution, Trails A and B and the Clock Drawing tests.26 We were also interested in the Montreal Cognitive Assessment (MoCA), Sum of Boxes in the Clinical Dementia Rating Scale (CDRSB) and the Alzheimer’s Disease Assessment Scale-Cognition Subscale, 13 tasks (ADAS-Cog 13) to get a deeper understanding of the participants’ cognitive profiles (see below).All participants underwent a comprehensive neuropsychological test battery. Here, t1 diagnosis made by the ADNI Clinical Core: cognitive normal (CN) , MCI  and Alzheimer’s disease . These classifications represent widely used cognitive and functional measures in clinical trials.27-29 Further information regarding diagnostic is available at http://adni.loni.usc.edu.Furthermore, we included participants’ t1 measurement in the analyses. To maximize the number of subjects, the second measurement was selected after an interval of 1.5 years ±12 months (t2).1 Further details on inclusion and exclusion criteria for participating in ADNI are available under http://adni.loni.usc.edu. In total, 153 participants for ADNI-GO/2 and 141 for ADNI 3 (only basic rsfMRI version) fulfilled our inclusion criteria. However, a large proportion had to be excluded mainly based on fMRI data quality  and abnormal CSF  values  temporally related to a rsfMRI acquisition (e.g. a participant’s screening MRI and baseline lumbar punction measurement). This measurement served as lity see . Thus, d) values .t1).http://adni.loni.usc.edu). The analyses presented here were approved by the local Ethics Committee of the University of Lübeck and carried out after ADNI’s recommendations including the approval of the manuscript before submitting to a journal.Each centre collecting data for ADNI provided an Institutional Review Board (IRB) approval and meets ADNI’s requirements. Informed consent was obtained from all ADNI participants  to compare baseline signals and the annual percentage signal change  between regions (NbM and EC as a within-subject factor) and CSF groups . Covariates such as age, sex, education, ADNI cohort and scanner manufacturer were included to adjust for different scan protocols and other potential scanner-related differences. All 2 × 2 (region × CSF group) mixed ANCOVAs were carried out in IBM SPSS statistics version 25 (SPSS) with type III sums of squares, and within-subject effects were interpreted without covariates.Z-scored data. Subsequently, the dependent overlapping correlations of NbM versus EC with CSF ratio were compared using cocor.32To better understand the relationship between disease status and functional MRI properties, CSF ratios (see section CSF biomarker) and functional MRI signals were considered in a linear regression model in SPSS. The functional MRI signal served as dependent variable and CSF ratio as independent variable. The regression was run with the ® R2020b with fitlm using the bisquare weight function with the default tuning constant. The same covariates as for the mixed ANCOVA were included in the model. Finally, the predictive models (NbM→EC and EC→NbM) were tested for each CSF group  and each functional property (fALFF and ReHo). The data were Z-scored before entering the analysis to ensure comparability of the APSC and baseline signal.To minimize the influence of outliers, especially in the APSC, robust regression models were carried out in MATLAB33 for fALFF and ReHo investigating whether CSF group assignment moderates the spread (NbM→EC versus EC→NbM) of functional degeneration. Here, CSF group was used as a dichotomous moderator variable. For the construction of products mean-centering was applied, and the heteroscedasticity consistent standard error HC3 (Davidson–MacKinnon) was applied.Moderation analyses were carried out in SPSS using the PROCESS macro34 was used to assess longitudinal APSC in fALFF and ReHo. It accounts for the days between both measurements and minimizes the influence of differences between both measurements within a subject.The following formulaBased on the CSF grouping strategy, we investigated how abnormal CSF and normal CSF groups performed in neuropsychological tests. For each test, one-way fixed effect ANOVAs were carried out with CSF group as factor and age, sex and education as covariates. As expected, the normal CSF group is less affected by cognitive impairment than the abnormal CSF group see .F = 7.943, P = 0.006, η2p = 0.112; F = 4.623, P = 0.035, η2p = 0.068; t(69) = 3.141, P = 0.002) but not EC (t(69) = 1.856, P = 0.068]. There was no main effect of region  and no significant group × region interaction  in APSC fALFF  = 9.437, P = 0.003; R²=0.031, F = 2.206, P = 0.142; Z = −1.94; P = 0.0262; 95% CI: −0.3429 to 0.0015). Additionally, we analysed both linear regressions independently for Aβ and pTau  = 4.19, P = 0.044; R²=0.09, F = 6.784, P = 0.011; R²=0.022, F = 1.556, P = 0.217; R²=0.003, F = 0.234, P = 0.63; In a next step, we used linear regressions on baseline fALFF values from NbM and EC, respectively, with CSF ratio as independent variable. It revealed a significant linear effect in the NbM . The other regression model EC→NbM did not show a significant effect , with a significant direct effect of NbM→EC , but no significant moderator effect , which is in line with the robust regression analysis.Finally, we performed two moderation analyses. The first model included baseline fALFF NbM as independent variable, fALFF EC APSC as dependent variable and CSF group as moderator. The model was statistically significant , which, again, is in line with the robust regression analysis.The second model included baseline fALFF EC as independent variable, fALFF NbM APSC as dependent variable and CSF group as moderator. The model was not statistically significant [The results for ReHo  and funcWe investigated the functional properties of the human NbM and EC in relation to the disease progression of Alzheimer’s disease based on longitudinal rsfMRI data and CSF markers of Aβ and pTau. With a focus on fALFF, our data provide evidence that spontaneous local brain activity in the NbM, but not EC, is reduced with CSF ratio, and, importantly, it predicts the APSC in the interconnected EC independently from proteinopathology. As such, our findings extend previous anatomical studies in humans and animals by providing novel physiological insights into the pathological staging model of Alzheimer’s disease suggesting the NbM as an early origin for subsequently affected brain regions possibly via a trans-synaptic mechanism.35 and the NbM is particularly vulnerable to the early accumulation of pTau36-38 and Aβ deposition.39 This may be due to the fact that cholinergic basal forebrain neurons have rather large axons and arbors reaching into the entire central nervous system with high metabolic demands for maintenance, reparation and transportation.40 At the same time, simply due to their sizes, they are more vulnerable to toxins,41 which may further promote disease progression.Local spontaneous brain activity, as quantified by fALFF, was reduced in the NbM at baseline in the abnormal CSF group , and the1 Our functional data support such a view by showing that the NbM’s baseline fALFF signal predicted the APSC in the EC  framework, which includes markers of Aβ, tau (T) and neurodegeneration (N).24 However, this requires a much larger sample of subjects than n = 71, since at least three groups would need to be included , and their distribution is typically unbalanced . Furthermore, the necessary cut-off values, which are often based on PET but in some studies CSF, are not well defined.24 Finally, we wanted to analyse our data as similar as possible to Fernández-Cabello et al. (2020) in order to be comparable.Finally, ADNI is a large multicentre study offering a rich and unique data set. This also means that our rsfMRI data come from different MR scanners, possibly leading to a bias in image quality and extracted signal. Therefore, we only included high-quality data based on comparable protocols and within-subject measurements from the same scanner. We also employed appropriate covariates in our statistical models, and differences in scanning parameters (e.g. slice order or number of volumes) were accounted for during preprocessing.Functional activity in the human basal forebrain scaled with proteinopathology and predicted the functional decline within the interconnected EC independent from CSF status. As such, our findings extend the pathological staging model of Alzheimer’s disease by giving novel insights into the functional properties of the underlying brain regions. From a more general perspective, fALFF appears to be a suitable marker to further investigate functional brain changes associated with the progression of Alzheimer’s disease.fcad262_Supplementary_DataClick here for additional data file.