Major brevican and neurocan cleavage products were measured in the CSF using semiquantitative immunoblotting. Total brevican and neurocan concentrations in serum and CSF did not differ between groups. The 60 kDa brevican fragment resulting from cleavage by the protease ADAMTS-4 was also found unchanged among groups. The presumably intracellularly generated 150 kDa C-terminal neurocan fragment, however, was significantly increased in ALS as compared to all other groups. This group also shows the highest correlation between cleaved and total neurocan in the CSF. Brevican and neurocan levels strongly correlated with each other across all groups, arguing for a joint but yet unknown transport mechanism from the brain parenchyma into CSF. Conclusively our findings suggest an ALS-specific pattern of brain ECM remodeling and may thus contribute to new diagnostic approaches for this disorder.The neural extracellular matrix (ECM) composition shapes the neuronal microenvironment and undergoes substantial changes upon development and aging, but also due to cerebral pathologies. In search for potential biomarkers, cerebrospinal fluid (CSF) and serum concentrations of brain ECM molecules have been determined recently to assess ECM changes during neurological conditions including Alzheimer’s disease or vascular dementia. Here, we measured the levels of two signature proteoglycans of brain ECM, neurocan and brevican, in the CSF and serum of 96 neurological patients currently understudied regarding ECM alterations: 16 cases with amyotrophic lateral sclerosis (ALS), 26 epilepsy cases, 23 cerebral small vessel disease (CSVD) patients and 31 controls. Analysis of total brevican and neurocan was performed  In the healthy adult human brain approximately 20-22% of the neural parenchyma represent extracellular space . This spECM structures create microcompartments for stem cell migration, diffusion processes, for the presentation of trophic factors to their cognate receptors and for synaptic and volume transmission  or ‘A Disintegrin and Metalloproteinase with Thrombospondin motifs’ (ADAMTS) enzymes. Like all lecticans, they have in common a dumbbell-like three-dimensional protein structure with hyaluronan-binding domains localized in the N-terminal globular parts and cell-surface- or glycoprotein-interacting domains in the C-terminus, such that proteolytic events within the central rod-like domain separate proteoglycan parts and loosen the ECM integrity . The majUnder aging and pathophysiological conditions the integrity and composition of the brain ECM undergo extensive changes, leading to a disturbed equilibrium of ECM production and deposition on one hand and ECM cleavage and clearance on the other hand . In humaFor amyotrophic lateral sclerosis (ALS), an adult-onset neurodegeneration of motor neurons, the picture is less clear, but studies in animal models revealed that in the surrounding of affected motor neurons a non-permissive microenvironment for regeneration is formed. In the lesion area in the adult spinal cord of a rat ALS model this microenvironment was shown to contain accumulations of chondroitin sulphate proteoglycans neurocan and versican . In the Sporadic and familial Cerebral Small Vessel Diseases (CSVDs), a spectrum disorder affecting blood-brain barrier and small vessel wall integrity, are also hypothesized to be linked to dysregulated ECM and its proteolytic fragmentation . CSVDs ain vivo and establish new biomarkers as a proxy for neural ECM reorganization. Thus far, serum and CSF biomarker studies have focused on traumatic brain injury (TBI), on vascular and neurodegenerative dementia and have shown lower CSF brevican, comprising its N-terminal peptides, and neurocan levels as well as decreased total brevican, but an increase of its C-terminal fragments in the patients’ serum  and total Tau (tTau) was determined either through ELISA ) or with an automated immunoassay ), following the manufacturer’s instructions  was calculated to assess blood-brain barrier (BBB) integrity, and the IgG-Index  was calculated to assess intrathecal immunoglobulin synthesis as a predictor for neuroinflammatory processes.In all patients and controls lumbar puncture (LP) and venous puncture (VP) have been conducted for diagnostic workup. For lumbar puncture patients were seated and 9 ml CSF were taken. In all samples, relevant CSF blood contamination was excluded visually/macroscopically and through microscopy of non-centrifugated CSF aliquots. After centrifugation, the pellet was separated to avoid the presence of cells in the final analysis samples. Within 20 min of LP and VP, CSF and serum samples were centrifuged at 4°C, aliquoted and stored at −80°C until brevican and neurocan analysis (see below). In ALS and CSVD, CSF markers of neuroaxonal damage and neurodegeneration were determined immediately after sample collection; they were not available in the epilepsy group. Neurofilament light chain (NF-L) was measured with commercially available ELISA . CSF The study was approved by the Ethics Committee of the Otto-von-Guericke-University Magdeburg (Sign 07/17). Patients donating biological material gave written informed consent in accordance with the Declaration of Helsinki. Data and material were handled in a coded fashion guaranteeing patient anonymity.For the quantitative measurement of total brevican and neurocan we used commercial anti-human brevican und anti-human neurocan ELISA kits, respectively  (ELH-BCAN-1 und ELH-NCAN-1). Serum samples were diluted 1:2 for brevican ELISAs and used undiluted for neurocan ELISAs. The dilution factor for CSF was 1:200 to 1:300 for anti-brevican ELISAs (to optimize the dilution for the detection range of the ELISA batches) and 1:10 for anti-neurocan ELISAs. ELISA measurements were essentially conducted according to the manufacturer’s instructions. In brief, pre-diluted CSF and serum samples were incubated in 96-well strip microplates pre-coated with monoclonal mouse anti-brevican (immunogen: aa 23-911) or polyclonal sheep anti-neurocan (immunogen: aa 23-1321). Samples were analyzed as triplicates or duplicates.To compensate for sensitivity differences in ELISA batches the calibrator concentration of the recombinant brevican or neurocan protein for the standard curve was adjusted according to OD values obtained .2SO4. OD values were measured in a VersaMax™ Tunable Microplate Reader  at 450 nm and at 650 nm for unspecific background subtraction. According to the manufacturer’s information intra- and inter-assay variability is below 10 and 12%, respectively.After 2.5 h incubation, 1:80 diluted biotinylated antibody against the target protein was added and incubated for 1 h, followed by a 45 min incubation with HRP-streptavidin (1:200 for brevican and 1:500 for neurocan) and a 30 min incubation with 3,3′,5,5′-tetramethylbenzidine in the dark. The reaction was stopped by adding 0.2 M HFollowing primary antibodies were used: rabbit anti-brevican “neo” (Rb399)  and peroxidase-conjugated AffiniPure donkey anti-sheep IgG (H + L) .For semiquantitative assessment of brevican and neurocan fragments undiluted CSF samples were incubated with chondroitinase ABC  at 1U/mg at 37°C for 30 min to digest chondroitin sulfate side chains and afterward solubilized in 5 × SDS loading buffer  and boiled at 95°C for 10 min. Samples were separated on 2,2,2-trichloroethanol (TCE)-containing stain-free 5–20% Tris-glycine SDS polyacrylamide gels under reducing conditions. Before Western blotting proteins were activated under UV light for 5 min. Protein transfer onto PVDF membranes  was performed according to standard protocols. To control for proper protein transfer UV images of the membranes were captured. After blocking for 1 h at RT in 5% horse serum  in TBS-T  Tween20, pH 7.6) membranes were incubated in primary antibodies overnight at 4°C. After washing three times with TBS-T for 10 min, secondary antibodies were added and incubated for 60 min at RT. After washing membranes again three times with TBS-T for 10 min, immunodetection was performed using an ECL Chemocam Imager . To improve semiquantitative comparability of optical density data from immunoblots we used a standard sample loaded on all gels as a reference to calibrate individual blot data and compensate for gel differences. Quantification of band intensities was done using NHI ImageJ software version 1.52a .post hoc comparisons were conducted using Tukey’s multiple comparison tests (Tukey’s HSD). For statistical comparisons within the CSVD (HA versus CAA) group, t-tests were calculated on age- and sex-corrected brevican and neurocan levels. For analyses of data uncorrected for sex and age, please see ρ) was used and p values were reported with Bonferroni correction for multiple testing. The level of significance was defined as p ≤ 0.05 and all tests were conducted two-tailed. Results were reported with Cohen’s d and partial eta squared (ηp2) as effect sizes. Statistical analysis was done using GraphPad Prism version 9  and SPSS Version 28 .For the analysis of brevican and neurocan levels non-parametric testing was used due to the sample characteristics, particularly the relatively small sample sizes of the ALS and CSVD groups. Because the overall groups were not matched for age and sex, a rank analysis of covariance was performed on brevican and neurocan levels following the protocol of F = 1.04, p = 0.38, ηp2 = 0.04, and neurocan total CSF concentration levels, F = 0.03, p = 0.99, ηp2 < 0.01; see F = 0.75, p = 0.53, ηp2 = 0.03, and for neurocan total serum levels, F = 1.84, p = 0.15, ηp2 = 0.07. No significant differences were measured within the CSVD subgroups (CAA versus HA) for brevican and neurocan total CSF concentration levels  and total serum concentration levels .The concentrations of total neurocan and total brevican were quantitatively assessed in patients’ body fluids with commercially available ELISAs. For all patient groups the mean values ± SD and the median values of raw data are given in ρ = 0.641, p < 0.001) which was observable in all sample groups  = 1.31, p = 0.28, ηp2 = 0.05) nor for the ratio of cleaved versus total brevican  = 1.81, p = 0.15, ηp2 = 0.06) . Across all groups, cleaved brevican fragment levels significantly correlated with total brevican concentrations  . Also, fF = 4.35, p < 0.01, ηp2 = 0.14. As shown in F = 8.48, p < 0.001, ηp2 = 0.25, with post hoc Tukey’s HSD tests again indicating significantly increased mean scores in the ALS compared to all other groups .Human neurocan fragments in the CSF appeared as 150 kDa C-terminal product and approx. 130kDa N-terminal fragment, which is in agreement with previous work . The ranTo detect correlations of the estimated CNS-derived proteoglycan concentrations in the patients’ body fluids with each other and with established biomarkers we performed a Spearman’s correlation analysis covering relevant diagnostic parameters obtained in the clinical work up . The serρ = 0.272, p = 0.01) and cleaved brevican levels  as well as for total neurocan  in the CSF we detected significant positive correlations with the age of the patients, which did not survive Bonferroni correction. Interestingly, CSF proteoglycan levels, except for cleaved brevican, also correlated strongly with the Alzheimer precursor protein fragment Abeta 1-40, a physiological brain-derived extracellular polypeptide . For cleaved as well as for cleaved/total brevican in the CSF, we observed significant correlations with CSF albumin and with the albumin quotient .For the total . In the CSVD sample, only total neurocan was significantly correlated with t-Tau values , and contrary to our expectations we did not find any correlation with NF-L levels in the patients’ CSF samples.Since ALS and CSVD are neurodegenerative disorders, we also explored the correlations with neurodegeneration markers NF-L and total Tau. As shown in As epilepsy can be caused by neuroinflammatory processes we checked our epilepsy cohort for the occurrence of encephalitis. There were no cases with acute, but 5 cases with putative subacute encephalitis. A comparison between these two subgroups revealed significantly lower amounts of CSF neurocan and neurocan-C in the encephalitis cases , howeverHere we show in a cohort of neurological patients that the CSF levels of the major proteolytic fragment of the perineuronal ECM proteoglycan neurocan, neurocan-C, are significantly higher in the ALS group as compared to controls, but also to epilepsy and CSVD patients. This effect became even more evident after normalizing concentrations of the cleaved to total core proteoglycan. The lack of significant findings from serum samples may be due to the more distal relation of serum to brain processes, but mostly due to the large dynamic range of plasma proteins and the huge intra- and inter-individual variability in serum protein composition. The low abundance of neurocan is another factor contributing to the lack of significant findings in serum samples.via the action of ECM-cleaving matrix metalloproteinases  also the passing rate across the BBB resp. the blood CSF barrier may affect the levels measured in body fluids. This assumption is supported by the observed correlation of cleaved versus total brevican in the CSF with the albumin quotient , arguingFinally, the glymphatic flux rate inside the brain parenchyma is supposedly a major factor in the regulation of the CSF composition. This assumption is supported by our finding that CSF proteoglycan levels largely correlate with the levels of Abeta 1-40, another neural polypeptide found in the extracellular space of the CNS and cleared by the glymphatic system , indicatThe neural ECM is a dynamic molecular filter for all cellular secretomes in the brain. It undergoes constant homeostatic remodeling and appears disorganized in several pathologies. So far, to our knowledge no other study investigated brevican and neurocan fragments as potential biomarkers for epilepsy, CSVD or ALS. However, in a series of biochemical or proteomic studies a Swedish group investigated brevican and neurocan already in a spectrum of other neurological conditions. In 2018 they demonstrated both brevican and neurocan CSF levels being lower after prophylactic cerebral radiotherapy of lung cancer patients, but only in cases without brain metastases . In TBI 42/40 values but a significant correlation between brevican, neurocan, and the brevican-interacting ECM glycoprotein tenascin-R   .With the help of a fragment-specific anti-brevican antibody, Jonesco and colleagues found in the serum of a highly heterogeneous group of patients with other dementias  slightly reduced amounts of total brevican but increased levels of the C-terminal brevican fragment derived from ADAMTS-4 cleavage, yielding a clearly increased ratio of cleaved over total brevican . In our This motoneuron disease is considered as non-cell autonomous, i.e., beyond the motor neurons also the surrounding glial cells are affected . To comeInterestingly, in ALS conditions two of the factors known to stimulate neurocan cleavage, namely EGF and TGFbeta  are affep = 0,0067; −0,39-fold change) in the CSF samples as compared to healthy control samples  fragments exerting signaling functions which may be different from the role of the uncleaved ECM components .Although transport modalities for brain-derived matrikines into the CSF and into the serum are still fully unclear, to study them under pathophysiological conditions could be of interest both from a clinical but also from a cell biological perspective.Further experiments are necessary to confirm these findings. in an independent sample and to clarify the clinical value.The statistical power from the relatively small numbers of individuals involved was felt to be too low to draw firm conclusions with regard to the suitability and disease specificity as bona fide biomarkers, however, our results are promising and deserve further validation in larger cohorts to unravel the full potential in the clinical work up, not least to come closer to the pathophysiological mechanisms behind our observations.Taken together, our experiments suggest that – beyond association with clinical phenotypes – the CSF levels of the neural proteoglycans investigated here seem to be differentially regulated: while total brevican and neurocan as well as the C-terminal fragment of neurocan appear to be largely controlled by the general glymphatic flux, the 60kDa brevican fragment seems to be rather controlled by the BBB function.From our work we conclude that proteolytic cleavage products of brain-derived perineuronal ECM molecules, such as neurocan fragments, may allow insights into the integrity of the brain’s extracellular environment, being potent CSF indicators for the actual proteolytic ECM fragmentation activity in the CNS tissue, especially if normalized to the concentration of total core proteoglycans. In the clinic, their correlation with established markers for neurodegeneration or BBB leakage may pave the way to a more differentiated diagnosis of neurological disorders, based on body fluids. Our results suggest to include the ratio of cleaved to total neurocan into informative biomarker panels to improve the discriminatory power between disorders.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethics Committee of the Otto von Guericke University Magdeburg. The patients/participants provided their written informed consent to participate in this study.CIS, EG and SS conceptualized the study. SS, CG, FS and SV assessed the clinical phenotypes and provided patient samples. CG provided the clinical data. WH and LH performed the experiments and designed the figures. CS and WH performed data analysis. CIS and EG designed the experiments and wrote the draft. All authors revised and finalized the manuscript and approved the submitted version.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.


Cerebrospinal fluid (CSF) p-tau235 is a novel biomarker highly specific of Alzheimer’s disease (AD). However, CSF p-tau235 has only been studied in well-characterized research cohorts, which do not fully reflect the patient landscape found in clinical settings. Therefore, in this multicentre study, we investigated the performance of CSF p-tau235 to detect symptomatic AD in clinical settings and compared it with CSF p-tau181, p-tau217 and p-tau231.n=212) and BIODEGMAR cohort . Patients were classified by the syndromic diagnosis  and their biological diagnosis .CSF p-tau235 was measured using an in-house single molecule array (Simoa) assay in two independent memory clinic cohorts: Paris cohort . CSF p-tau235 was pronouncedly increased in the A+T+ profile group compared with A−T− and A+T− groups . Moreover, CSF p-tau235 demonstrated high diagnostic accuracies identifying CSF amyloidosis in symptomatic cases (AUCs=0.86 to 0.96) and discriminating AT groups (AUCs=0.79 to 0.98). Overall, CSF p-tau235 showed similar performances to CSF p-tau181 and CSF p-tau231 when discriminating CSF amyloidosis in various scenarios, but lower than CSF p-tau217. Finally, CSF p-tau235 associated with global cognition and memory domain in both cohorts.High CSF p-tau235 levels were strongly associated with CSF amyloidosis regardless of the clinical diagnosis, being significantly increased in MCI Aβ+ and dementia Aβ+ when compared with all other Aβ− groups (Paris cohort: CSF p-tau235 was increased with the presence of CSF amyloidosis in two independent memory clinic cohorts. CSF p-tau235 accurately identified AD in both MCI and dementia patients. Overall, the diagnostic performance of CSF p-tau235 was comparable to that of other CSF p-tau measurements, indicating its suitability to support a biomarker-based AD diagnosis in clinical settings.The online version contains supplementary material available at 10.1186/s13195-023-01201-0. Thesid (CSF)  often by1–42/40 can be detected [Recently, the field of fluid biomarkers in neurodegeneration has undergone a remarkable expansion, particularly with regard to biomarkers measuring different variants of tau protein. This has resulted in a wide range of novel p-tau biomarkers, measured with different methods and platforms  –11. Diffdetected . Interesdetected . With clcontinuum and a preclinical AD cohort. We demonstrated that CSF p-tau235 displays a statistically equal performance to that of CSF p-tau217 and p-tau231 when discriminating cognitively impaired CSF Aβ-positive cases from cognitively impaired CSF Aβ-negative cases. However, the cases enrolled in that study belonged to a well-characterized research cohort, which does not fully reflect the patients from daily clinical practice including atypical AD, other dementia and comorbidities. Therefore, evaluating the performance of CSF p-tau235 in the routine practice of a memory clinic is much needed, since in these settings patients present with a higher heterogeneity in demographics, comorbidities and disease presentations [In order to further characterize this novel p-tau biomarker and contextualize its performance with other CSF p-tau species, we previously compared CSF p-tau235 with CSF p-tau217 and p-tau231 in a well-characterized research cohort including the AD ntations –25. As aTo this end, we measured this novel p-tau biomarker in two independent memory clinic cohorts, compared its performance with N-terminal directed Simoa immunoassays targeting CSF p-tau181, p-tau217 and p-tau231, and assessed its relation with cognitive status.n=21), non-AD mild cognitive impairment , AD-MCI (n=40), AD dementia (n=75) and non-AD dementia (n=31). Non-AD dementia patients encompassed patients with dementia with Lewy bodies , frontotemporal dementia , vascular cognitive impairment and dementia  and Creutzfeldt Jakob disease (n=1). Patients underwent a thorough clinical examination involving personal medical and family histories, treatment, neurological examination, extensive neuropsychological assessment, APOE genotyping, brain magnetic resonance imaging (MRI) extensive neuropsychological evaluation, MRI, APOE genotyping, blood and CSF analysis and fluid sampling for collection (blood and CSF). The diagnosis for each patient was made during multidisciplinary consensus meetings  considering results of validated CSF biomarkers and according to clinical diagnostic criteria for AD dementia [continuum [) included patients with psychiatric disorder, sleep apnea, or systemic disease. Non-AD MCI presented with normal CSF biomarkers or suspected non-Alzheimer pathophysiology . Included SCD participants were individuals with several years of clinical follow-up for a clinical complaint, presenting with normal cognitive testing and no abnormalities at imaging and CSF examinations [Paris cohort enrolled a total of 212 patients who had undergone CSF analysis at the Centre of Cognitive Neurology at Lariboisière Fernand-Widal University Hospital between March 2014 and December 2019, including participants with subjective cognitive decline , MCI (n=74) and, for subjects with moderate to severe cognitive impairment, possible AD dementia (n=11), probable AD dementia (n=43), LBD (n=2), extrapyramidal syndrome (n=2), vascular cognitive impairment and dementia , progressive supranuclear palsy , corticobasal syndrome (n=4), behavioral variant of FTD , primary progressive aphasia  and cerebral amyloid angiopathy .The BIODEGMAR cohort is an observational longitudinal study that enrolls patients with neurodegenerative diseases visiting the Cognitive Decline and Movement Disorders Unit of Hospital del Mar . The procedures of the BIODEGMAR study include extensive neuropsychological evaluation, MRI, mination , 32. In 1-42/40, Supplementary Table 1–42/40 and p-tau181, respectively (Lumipulse®) into A−T−, A+T− and A+T+ . The clinical diagnoses included in each group are available in Supplementary Tables Patient data from the Paris cohort and the BIODEGMAR cohort was examined using two stratification criteria: (i) according to clinical syndrome  and CSF amyloid status  and the BIODEGMAR cohort . In the Paris cohort, memory domain scores were evaluated using total immediate and delayed recall of Free and Cued Selective Reminding Test (FCSRT), and the delayed matching-to-samples 48 test (DMS 48) for visual memory testing. Executive function was assessed using forward and backward digit span, frontal assessment battery and letter and animal fluencies. The language domain was evaluated using the Dénomination Orale 80 test, a naming test. In BIODEGMAR cohort, memory domain was evaluated using total immediate and delayed recall of FCSRT and the Memory Impairment Screen; executive functions, with backward digit span, TMT A and B; language domain, with the Boston naming test. Z-scores were computed from the control group scores as a reference. Domain scores were obtained by averaging z-scores of the individual tests available results within that domain and the global cognition score by averaging the 3 domains’ z-scores.All included participants were evaluated using the Mini-Mental State Examination (MMSE). Detailed cognitive assessment was available for a subsample of participants in both the Paris cohort  were measured using the clinically validated Lumipulse® G1200 assay (Fujirebio) [in-house Simoa assays. CSF p-tau181 and p-tau217 measurements were previously reported [1–42/40 ratio, p-tau181 and t-tau) were measured at Laboratori de Referència de Catalunya (LRC) with Lumipulse G600II (Fujirebio). Cutoff values for biomarkers and their ratios  were previously defined in the CORCOBIA study [in-house Simoa assays and were previously reported [In the Paris cohort, core AD CSF biomarkers (Aβjirebio) . Biomarkreported  and wereIA study . Biomarkreported .r) and intermediate precision (%CVRw) values for both cohorts were ˂15%. Further details on assay specifications and validation can be found elsewhere [CSF p-tau235 measurements were performed at the Clinical Neurochemistry Laboratory at the University of Gothenburg using a Simoa HD-X instrument (Quanterix), and all samples were blinded. Prior to the analysis, samples were allowed to thaw at room temperature for 45 min. Thawed samples were subsequently vortexed for 15 s, after which they were plated and diluted 1:2 using Tau2.0 sample assay diluent (Quanterix). Due to sample volume availability, CSF samples from the Paris cohort were run in singlicates, while BIODEGMAR cohort samples were run in duplicates. An eight-point calibration curve was generated using recombinant full-length GSK-3β-phosphorylated tau-441  and run in duplicates. Two internal quality controls  were run at the beginning and end of each plate, also in duplicates. Repeatability (%CVhttps://www.r-project.org/). Boxplots show the median and interquartile range (IQR), with individual data points for all participants always shown. Statistical analyses were performed using parametric or non-parametric methods when appropriate, based on the normality or not of the data. Group comparisons between two categories were performed using a t-test and Mann-Whitney U test. All comparisons of multiple groups were tested with a one-way ANOVA adjusted for age and sex followed by Bonferroni-corrected post-hoc pairwise comparisons. Adjusted P-values for covariates are indicated as PADJ. For each of the CSF p-tau biomarkers, accuracy for binary outcomes was determined using receiver operating characteristics (ROC) analysis and area under the curve (AUC). AUC values are accompanied by the 95% confidence interval, abbreviated as CI95%. Performance comparison between different p-tau biomarkers was analysed using DeLong test . Spearman’s rank correlation (rS) was used to determine associations between biomarkers. Association of CSF p-tau biomarkers with global and domain-specific cognitive z-scores was studied with linear regression models adjusted for age, sex and level of education.Statistics were performed using SPSS  and Scistat . Graphs were generated using GraphPad PRISM  and R version 4.1 . Demographics and stratification in syndromic groups  are presented in Table P<0.0001). There was a weak yet significant association with age in both cohorts . There were no sex differences between groups . In both cohorts, CSF p-tau levels were significantly higher in APOE ε4 carriers . As expected, there were significant differences in MMSE scores across groups in both cohorts (P <0.0001), which, as compared with CU, gradually decreased in MCI and further in patients with dementia.A total number of 387 participants were included in this study, which belong to two independent clinical cohorts   , but no statistical differences were found between MCI Aβ+ and CU Aβ+. No significant differences were found between CU Aβ− and CU Aβ+, likely due to small sample sizes , although higher CSF p-tau235 levels in CU Aβ+ were noticeable  and MCI Aβ+ from MCI Aβ−   and CSF p-tau231 . In contrast, lower accuracies were observed when compared with CSF p-tau217   and MCI Aβ+ from MCI Aβ−   and CSF p-tau231 . On the other hand, the accuracy of CSF p-tau235 distinguishing MCI Aβ+ from MCI Aβ− approached significance compared with CSF p-tau181 , while matched CSF p-tau231  . This was followed by a prominent increase between A+T− and A+T+ (PADJ <0.0001). CSF p-tau235 concentration was also significantly higher in A+T+ when compared with A−T− (PADJ <0.0001)   , similar to CSF p-tau231 , but lower than both CSF p-tau181  and CSF p-tau217   and A−T− from A+T+ . In both scenarios, CSF p-tau235 performance was statistically equal to that of CSF p-tau181 , CSF p-tau217  and CSF p-tau231   Fig. a. CSF p-A−T− vs A+T−=0.79, CI95%=0.68–0.87) as CSF p-tau181  and CSF p-tau231 . The accuracy of CSF p-tau235 when discriminating A+T− and A+T+  statistically matched that of CSF p-tau181  and slightly outperformed that of CSF p-tau231 . Finally, CSF p-tau235 showed a nearly perfect accuracy discriminating A−T− and A+T+ , same as that observed for CSF p-tau181  and CSF p-tau231  . When compared with other p-tau species  and CSF p-tau 217 , but equal to CSF p-tau231   matched the performance of both CSF p-tau181  and CSF p-tau231  . The association remained significant after further adjustment on Aβ1–42/40 ratio .In both cohorts, MMSE score was associated with CSF p-tau235 after adjustment on age, sex and level of education  and BIODEGMAR cohort (n=139). The cross-sectional associations of CSF p-tau235 levels and other CSF p-tau measurements with global and domain-specific cognition are shown in Fig. PADJ =0.046; BIODEGMAR β=−0.363, PADJ <0.0001), similar to all CSF p-tau measurements but CSF p-tau217. Regarding specific cognitive domains, the strongest association was found with memory impairment, both for CSF p-tau235 and other p-tau species . A weak association was also found with executive domain function but only in BIODEGMAR .Neuropsychological assessment was available for a subset of patients in the Paris  CSF p-tau235 is strongly associated with CSF amyloidosis, regardless of clinical presentation; (ii) high levels of CSF p-tau235 in cognitively impaired patients are highly indicative of MCI or dementia of the AD type; (iii) the main increase in CSF p-tau235 levels occurs between A+T− and A+T+, irrespective of the presence of symptoms or not; (iv) CSF p-tau235 displayed overall the same diagnostic performance as CSF p-tau181 and p-tau231, but a slightly lower diagnostic performance compared with CSF p-tau217; and (v) CSF p-tau235 levels associate with global and specific-domain cognitive decline.continuum using two research cohorts, demonstrating its high specificity for AD and its comparable performance to other CSF p-tau species [1–42/40) [In a previous study, we described CSF p-tau235 across the AD  species . However1–42/40) , CSF p-t1–42/40) . Moreove1–42/40) . Further(i.e. A+T+ cases), regardless of these cases being symptomatic, as shown here, or asymptomatic, like previously reported [continuum AD, thus rendering a lower discrimination between A+T− and A+T+ groups [CSF p-tau235 followed an increasing trajectory across AT groups, which was characterized by a minor increment between A−T− and A+T−, followed by a prominent increase from A+T− to A+T+. These differences across AT groups are similar to those previously reported in preclinical AD , despitereported . CSF p-treported , 35. CSFreported . On the + groups . Finally+ groups . The samHigher CSF p-tau235 levels were associated with lower global cognition and memory function in both cohorts, which had not been previously investigated. CSF p-tau181 and CSF p-tau217 have been reported to associate with global cognition and memory impairment cross-sectional and longitudinally, with p-tau217 showing stronger associations with cognitive decline . In our continuum (such as p-tau231 and p-tau217) [. A limited number of patients in non-AD dementia group also warranted further studies.The strengths of this study include the use of two independent memory clinic-based cohorts, allowing us to validate and confirm our findings in two different clinical settings. Secondly, both cohorts had clinically validated biomarker measurements available, which enabled the detailed stratification of participants and the subsequent investigation of CSF p-tau235 in syndromic or AT groups. Moreover, the studied cohorts included measurements of other novel p-tau species, specifically N-terminal directed CSF p-tau181, p-tau217 and p-tau231 , making possible a head-to-head comparison between different p-tau residues. However, this study does not go without limitations. CSF p-tau217 measurements were not available in the BIODEGMAR cohort, and therefore, the biomarker comparison with CSF p-tau235 could only be explored in the Paris cohort. Another limitation is that CU Aβ+ cases were limited to the BIODEGMAR cohort, and their sample size was rather small. Considering that CSF p-tau235 increases late during preclinical AD , it is e-tau217)  would prcontinuum. Future studies will aim to elucidate whether CSF p-tau235 can predict cognitive decline in longitudinal samples and attempt to measure p-tau235 in blood samples.In conclusion, our memory-clinic-based study including two well-characterized cohorts brings further evidence that p-tau235 is a novel and specific CSF biomarker for AD diagnosis, both at MCI and dementia stages. Comparison with other CSF p-tau species, including p-tau181, p-tau217 and p-tau231, supports that CSF p-tau235 should be suitable for use in clinical settings. In addition, significant association with cognitive decline adds an argument to its relevance as a clinical tool to identify and monitor CSF amyloidosis along the whole AD Additional file 1: Supplementary Figure 1. CSF levels of p-tau235 across clinical diagnosis. Supplementary Figure 2. CSF levels of p-tau235 in Aβ+ and Aβ- cases. Supplementary Figure 3. CSF p-tau235 diagnostic performance discriminating Aβ+ from Aβ- cases. Supplementary Table 1. Clinical diagnosis included in each syndrome group in Paris cohort. Supplementary Table 2. Clinical diagnosis included in each syndrome group in BIODEGMAR cohort. Supplementary Table 3. CSF AD biomarkers cut-offs for BIODEGMAR and Paris Cohort. Supplementary Table 4. Accuracies of CSF p-tau181, p-tau217, p-tau231 and p-tau235 when identifying CSF amyloidosis in dementia and MCI cases in Paris and BIODEGMAR cohort. Supplementary Table 5. Clinical diagnosis included in each AT group in Paris cohort. Supplementary Table 6. Clinical diagnosis included in each AT group in BIODEGMAR cohort. Supplementary Table 7. Accuracies of CSF p-tau181, p-tau217, p-tau231 and p-tau235 when discriminating AT groups in Paris and BIODEGMAR cohort. Supplementary Table 8. Accuracies of CSF p-tau181, p-tau217, p-tau231 and p-tau235 when discriminating Aβ- from Aβ+ in Paris and BIODEGMAR cohort.


Dear Editorsliving-donor lung transplant is noteworthy because it was performed in a major incompatible state (wherein a recipient with type O blood receives a transplant from a donor with type A/B/AB). To date, only one major incompatible blood type lung transplant (MIBTLT), from a brain-dead donor, has been reported in Germany  announced a successful incompatible blood type lung transplant from living donors  . HoweverMIBTLT procedure may be due to the freshness of the living-donor lungs. We need to know how long after brain-death the lung was transplanted in the German operation. Studying what immunological and histological events occur in those 8 hours of clinical preservation is necessary.The clinical preservation time limit for lungs seems to be roughly 8 h. It is important to note that the clinical preservation time was absent in this case. The success of the ex-vivo lung perfusion (EVLP). However, it appears that the main purpose of EVLP is to prolong clinical preservation time. EVLP trials have so far provided little insight into the immunological mechanisms that make MIBTLT possible.We are fully aware of the Toronto group’s attempts at MIBTLT from a brain-dead lung donor, in Germany in 2007    reportedEVLP technique requires a circuit with multiple, complex components, depending on the device used. It commonly includes some form of drainage from the left atrium, chamber reservoir, centrifugal pump, membrane gas/heat exchanger, filtered gas. It commonly includes some form of drainage from the left atrium, chamber reservoir, centrifugal pump, membrane gas/heat exchanger, filtered gas line for deoxygenation, leucocyte filter, inflow cannula into the pulmonary artery and a ventilator connected to the trachea  becomes difficult. The psychological pressure that is placed on potential donors (who are often family members) will undoubtedly increase. In the case of living-donor transplantation, voluntariness is essential, since donors are healthy individuals. Strict regulations need to be established in each country to prevent coercion of potential donors.Sixth, ethical issues: Serious ethical issues emerge when considering the norm . If MIBTMIBTLT being performed in living-donor lungs, where a favorable outcome was achieved. This report is significant because the success of ABO MIBTLT  may increase treatment selection, thereby reducing potential organ shortages.Lastly, this is the first case of ABO


Due to the blood-brain barrier, plasma is not an ideal source to evaluate the genetic characteristics of central nervous system tumors. Thus, cerebrospinal fluid (CSF) is becoming an alternative biopsy type to evaluate the genetic landscape of intracranial tumors. We aimed to explore the genetic profiles of CSF-derived circulating tumor DNA (ctDNA) to predict intracranial tumor responses and monitor mutational evolution during the treatment of non-small cell lung cancer (NSCLC) patients with brain metastases.We conducted a prospective study of 92 newly diagnosed NSCLC patients with brain metastases. Paired CSF and plasma samples were collected at baseline, 8 weeks after treatment initiation, and disease progression. All samples underwent next-generation sequencing of 425 cancer-related genes.P=0.003). Moreover, patients with ≥ 50% reductions in the concentrations of CSF ctDNA after 8 weeks of treatment had significantly longer intracranial progression-free survivals (PFS) than patients with < 50% reductions in CSF ctDNA concentrations . A ≥ 50% reduction in CSF ctDNA concentrations had better concordance with radiographic intracranial tumor responses than plasma. A ≥ 50% reduction in plasma ctDNA concentrations was also associated with longer extracranial PFS . Based on clonal evolution analyses, the accumulation of subclonal mutations in CSF ctDNA was observed after 8 weeks of treatment. The clonal mutations that remained in more than 80% in CSF after 8 weeks also predicted shorter intracranial PFS .At baseline, the positive detection rates of ctDNA in CSF, plasma, and extracranial tumors were 63.7% (58/91), 91.1% (82/90), and 100% (58/58), respectively. A high level of genetic heterogeneity was observed between paired CSF and plasma, while concordance in driver mutations was also observed. A higher number of unique copy number variations was detected in CSF-ctDNA than in plasma. ctDNA positivity of CSF samples at baseline was associated with poor outcomes (HR=2.565, CSF ctDNA exhibited unique genetic profiles of brain metastases, and dynamic changes in CSF ctDNA could better predict intracranial tumor responses and track clonal evolution during treatment in NSCLC patients with brain metastases.ClinicalTrials.gov identifier: NCT 03257735.The online version contains supplementary material available at 10.1186/s12916-022-02595-8. Brain metastases occur in nearly 30–40% of lung cancer patients and always result in poor clinical outcomes . The treCirculating tumor DNA (ctDNA) is a promising biomarker to characterize the mutational profiles of tumors. Through real-time dynamic monitoring, ctDNA can also be used to trace tumor evolution and treatment responses, and identify resistance mechanisms , 6. HoweWe conducted this prospective cohort study to compare the genetic landscapes of paired CSF and plasma, and sought to trace intracranial tumor evolution and treatment responses through serial ctDNA sequencing of paired CSF and plasma samples in non-small cell lung cancer (NSCLC) patients with brain metastases.This prospective study  was designed and performed at the Sun Yat-sen University Cancer Center . Main inclusion criteria included following: (1) NSCLC newly diagnosed by histopathology; (2) brain metastases confirmed by enhanced brain MRI at primary diagnosis, with at least one intracranial lesion whose longest diameter was > 5 mm; (3) treatment-naïve ; (4) brain metastases were asymptomatic or responding to corticosteroid treatment, followed by systemic therapy as the first-line treatment; and (5) had no contraindication for lumbar puncture. All patients meeting the inclusion criteria were enrolled consecutively between January 2017 and December 2020 at our institution. For first-line treatment, patients received systemic therapies based on their mutational characteristics. Paired CSF and blood samples with or without primary extracranial tumor samples were collected at baseline (before treatment), 8 weeks after the initiation of first-line treatment, and at the time of disease progression. At each time point, 5 ml of CSF and 8 ml of blood were collected, and radiographic evaluations were performed within one week . Radiographic evaluations were then performed at approximately 8-week intervals until tumor progression. The clinical response was assessed according to the Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1 by a radiologist who was blinded to the patients’ clinical information. This study was approved by the ethics committee of the Guangdong Association Study of Thoracic Oncology . All patients provided written informed consent to participate in the study and provide samples for tumor genetic profiling.https://github.com/lh3/bwa/tree/master/bwakit). PCR duplicates were removed by Picard (available at: https://broadinstitute.github.io/picard/). The Genome Analysis Toolkit (GATK) (https://software.broadinstitute.org/gatk/) was used to perform local realignments around indels and base quality reassurance. Single nucleotide polymorphisms (SNPs) and indels were analyzed by VarScan2 [CSF and whole blood samples were collected in cell-free DNA BCT tubes . Within 24 h of CSF and blood collection, the cellular fraction was removed by two-step centrifugation at 4°C . The white blood cells were used for genomic DNA extraction  as the germline controls. Samples were stored at -80°C until further processing. CtDNA was extracted using the QIAamp Circulating Nucleic Acid Kit (Qiagen), as previously reported . For patVarScan2  and HaplVarScan2 . The medThe ctDNA levels were calculated as previously described , 20: maxTo evaluate the mutations that were consistently identified in the paired samples (sample A and sample B), the overlapping genes and unique genes from each sample were illustrated using a Venn diagram. Consistency was calculated using the following equation:Consistency was analyzed for all mutant-positive samples. When considering the factors of the somatic mutations and driver gene variants, consistency was analyzed after removing the copy number variants (CNVs) or non-driver gene variants from the samples.At baseline, a mutation was considered clonal if its VAF was more than 25% of the maxVAF, and it was defined as subclonal if it was below this threshold . Newly ap-values of less than 0.05 were considered statistically significant.Survival data were analyzed using Kaplan-Meier curves and Cox proportional hazard regression analyses. Comparisons of the continuous variables were made using an unpaired two-tailed t-test, while comparisons of the categorical variables between groups were performed using the chi-squared test or Fisher’s exact test. The concordance of genomic alterations between CSF and plasma, and concordance of dynamic changes in ctDNA and radiographic response was assessed using Cohen’s kappa coefficient. All statistical analyses were performed using R software (version 3.5.3). Two-tailed The study flow chart is shown in Fig. In total, 91 CSF samples and 90 plasma samples were collected from 92 patients at baseline (before treatment), and 58 patients had paired extracranial tumor tissues at baseline (38 primary lung tumor tissues and 20 lymph node tissues). Then, 49 CSF samples and 51 plasma samples were collected from 51 patients 8 weeks after the initiation of first-line treatment. Seven CSF samples and nine plasma samples were collected from nine patients at disease progression  of CSF samples, 91.1% 82/90) of plasma samples, and 100% (58/58) of extracranial tumor tissues   .Patients with ctDNA detected in the baseline CSF samples had significantly shorter overall survivals than patients without ctDNA detected in CSF samples at baseline  in CSF ctDNA and 51.8% (29/56) in plasma ctDNA, with a concordance rate of 87.5% between the CSF and plasma samples (Cohen’s kappa coefficient = 0.751). The mutation rate of TP53 was 48.2% (27/56) in CSF ctDNA and 60.7% (34/56) in plasma, with a concordance rate of 76.8% (Cohen’s kappa coefficient = 0.539). KRAS G12C was the predominant alteration in KRAS and demonstrated a concordance rate of 83.1% (Cohen’s kappa coefficient = 0.677) between CSF and plasma samples , and the majority of CNVs were unique to CSF samples. EGFR copy number gain was the most frequent CNV and was identified in 14.3% (8/56) of CSF and 7.1% (4/56) of plasma samples. Copy number loss of STK11, as well as the copy number gain of multiple genes, including NTRK1, NKX2-1, SMO, and CDK6 were identified exclusively in CSF ctDNA  than in plasma    Fig. A. The ex50) Fig. B.Fig. 4DP = 0.008) . In multivariate analyses, CSF ctDNA response remained a favorable predictive factor for intracranial PFS, regardless of treatment regimens  ; however, the finding was not statistically significant   of CSF samples, and in 57.1% (16/28) of plasma samples . However, the maintenance of clonal mutations in post-treatment plasma samples was not a significant prognostic factor for extracranial . Table S2. Univariate and multivariate Cox regression analyses of overall survival based on baseline variables (N = 92). Table S3. The comparison of clinical characteristics in patients with CSF ctDNA response and non-response after treatment (N = 25). Table S4. Univariate and multivariate Cox regression analyses of intracranial progression free survival based on baseline variables (N = 25). Table S5.The comparison of clinical characteristics in patients with plasma ctDNA response and non-response after treatment (N = 44).


With the continuous advancements of urbanization, the demand for power cables is increasing to replace overhead lines for energy transmission and distribution. Due to undesirable scenarios, e.g., the short circuit or poor contact, the cables can cause fire. The cable sheath has a significant effect on fire expansion. Thus, it is of great significance to carry out research on flame-retardant modification for cable sheath material to prevent fire accidents. With the continuous environmental concern, polyolefin (PO) is expected to gradually replace polyvinyl chloride (PVC) for cable sheath material. Moreover, the halogen-free flame retardants (FRs), which are the focus of this paper, will replace the ones with halogen gradually. The halogen-free FRs used in PO cable sheath material can be divided into inorganic flame retardant, organic flame retardant, and intumescent flame retardant (IFR). However, most FRs will cause severe damage to the mechanical properties of the PO cable sheath material, mainly reflected in the elongation at break and tensile strength. Therefore, the cooperative modification of PO materials for flame retardancy and mechanical properties has become a research hotspot. For this review, about 240 works from the literature related to FRs used in PO materials were investigated. It is shown that the simultaneous improvement for flame retardancy and mechanical properties mainly focuses on surface treatment technology, nanotechnology, and the cooperative effect of multiple FRs. The principle is mainly to improve the compatibility of FRs with PO polymers and/or increase the efficiency of FRs. Power cables are important equipment for energy transmission and are closely related to system security . Due to At present, the materials used for cable sheath are mostly organic polymers, including PVC, polyethylene (PE), polypropylene (PP), ethylene vinyl acetate (EVA), etc., of which PE includes high-density polyethylene (HDPE), low-density polyethylene (LDPE), and linear low-density polyethylene (LLDPE) ,5. PVC i2, CH4, CO, etc.) and thus intensify the combustion. Then a series of chain reactions occur, as shown in It is necessary to know about the interaction between the PO polymer and the ignition source before preparing an effective flame retardant. (i)Covering effect: When heated, some FRs can form a non-flammable protective layer, thereby blocking the two elements, i.e., oxygen and heat, necessary for combustion.(ii)2 and water vapor, when heated, and this can reduce the oxygen concentration around the polymers, thereby inhibiting the combustion process.Dilution effect: Some FRs can release incombustible gases, such as CO(iii)Endothermic effect: Some FRs can undergo a decomposition reaction to absorb a large amount of heat, leading to a cooling effect on the polymer.(iv)Inhibition effect: The thermal decomposition of some FRs will generate a large number of radicals, which can combine with the reactive radicals released by the polymer matrix to interrupt the process of the chain reaction.The corresponding basic theories of flame inhibition mode of action can be summarized as follows :(i)CoverTherefore, the action of FRs is mainly to interfere with the five stages of the combustion process according to these four modes of action, so as to reduce the spread of fire. This paper summarizes the research progress related to the halogen-free FRs, i.e., inorganic FRs, organic FRs, and IFR, which are used to cooperatively improve the flame-retardant properties and mechanical performance of PO matrix in recent years.2O3, have high specific surface energy and strong adsorption capacity. They can cover the outer surface of the PO polymer, thus forming a protective barrier to suppress smoke and slow the spread of fire [As FRs for PO materials, aluminum hydroxide (ATH) and magnesium hydroxide (MH) share the similar flame-retardant principle. The reaction formula is shown in  of fire .However, MH or ATH often require a large number of addition (50–60 wt.%) to achieve good flame-retardant properties. Furthermore, the polarity difference between hydroxide FRs and PO leads to poor compatibility, which seriously reduces the mechanical properties of the composites, such as elongation at break and tensile strength ,14.2+ (MH-PZPN-Ni) with core–shell structure by effectively grafting γ-aminopropyl triethoxysilane (KH-550), hexachlorocyclotriphosphazene (HCCP), 1-(2-Aminoethyl) piperazine (AEP), and Ni2+ on the surface of MH through the strategy of layer-by-layer assembly and method of Ni2+ chelation. The synthesis principle is shown in To improve the flame retardancy and mechanical properties of PO composites, researchers have proposed the method of surface modification for MH/ATH, which can change the surface properties of the metal hydroxide to improve its interfacial adhesion with PO polymers ,16. Sila2. They prepared EVA/ATH and EVA/ATH/GnPs/MoS2 samples through the method of melt blending, in which the EVA/ATH composite was recorded an LOI of 26% and a V-2 rating in UL-94 test. The elongation at break and tensile strength were 359% and 12.6 MPa. After the introduction of GnPs and MoS2, the LOI of the composite was 29.5%, and the UL-94 test reached V-0 rating. The elongation at break and tensile strength were 448% and 21.5 MPa, respectively. Nanoplatelets appeared to increase resistance to deformation and improve the modulus at break as proposed in 2 on the mechanical performance of EVA/ATH composites. The test results showed that GNPs had an adsorption effect on the polymer molecular chain. When the polymer is under tension, this adsorption can prevent the breaking of the molecular chain, thereby enhancing the mechanical properties of the polymer.In addition, researchers have found that nano-hydroxide obtained by ultra-fabrication shows the characteristics of small volume size, low filling amount, and large specific surface area. Moreover, nanoscale particles are easier to disperse uniformly in the PO matrix; thus, they have less negative impact on the mechanical performance of the polymeric material when used as FRs . Wang et3· at high temperatures, while RP or APP generates PO· and PO2·. These radicals will combine with each other and terminate the active radicals to achieve the purpose of interrupting and inhibiting the combustion of PO, thereby improving flame retardancy [3PO4), metaphosphoric acid (HPO3), and polymetaphosphoric acid decomposed from inorganic phosphorus FRs, will contribute to the dehydration of PO into carbon. This carbon layer can block the generation of new radicals by limiting the diffusion of oxygen in the combustion environment and protect the underlying polymer from further combustion. At the same time, thermal decomposition reactions will lead to the condensation of phosphoric acid and will release water to decrease the temperature of the matrix and dilute the concentration of combustible materials in the air, thus producing a flame-retardant effect [As one of the optimal alternatives for halogen-containing FRs , phosphotardancy . Meanwhit effect .However, the compatibility of RP with PO matrix is poor, and this adversely affects the mechanical performance of the PO composite. In addition, the low ignition point of RP will be a safety hazard in the application. These factors above lead to the restriction of the RP application.To solve this issue, RP should be treated before use. An effective method is to encapsulate the RP by using the so-called microencapsulation technology ,34. MicrMoreover, in order to make the polymer achieve higher flame-retardant properties, MRP often needs to be compounded with other FRs, e.g., nanocarbon materials and metal salts ,39. Chen2 as adjuvants to obtain PO composites with better flame retardancy and mechanical performance. After adding 30 wt.% AHP/MCA/MoS2 (18.7/9.4/2), the LOI value of EVA increased to the maximum value of 38.5%, and a V-0 classification was achieved in the UL-94 test. In addition, the elongation at break of the EVA composites increased to 654% after adding 2 wt.% MoS2.Up until now, microencapsulation has been the most frequently used technique to enhance the compatibility of inorganic phosphorus flame retardants with PO substrates, so as to obtain cable material with excellent flame-retardant and mechanical properties ,44. FurtIn summary, in order to achieve higher flame-retardant efficiency, the development of inorganic phosphorus FRs mainly focuses on the following aspects ,50,51: , glass fibers, silica gel and talcum powder, etc.Silicon FRs are divided into organic silicon FRs and inorganic silicon FRs. Inorganic silicone FRs have the characteristics of being non-toxic and having less smoke, low burning value, slow flame propagation, etc. . They in2 [However, the poor compatibility between inorganic silicon FRs and PO substrate will seriously damage the processability and mechanical performance of the composites. Therefore, how to improve the compatibility has become a key issue in the research of inorganic silicon FRs. Micronization  and surface treatment are common methods so far. Moreover, silicon FRs can be used with other additives to modify PO polymers for flame retardancy and mechanical properties . In gene2 ,55 and M2 ,57. Thus2 contains many advantages, including small particle size, large specific surface area, and good thermal stability. As an adjuvant for other FRs, it can form a protective layer of silicon on the surface of the material to enhance the flame-retardant effect [SiOt effect . The prot effect .2, including fused silica, fumed silica, and silica gel. Kashiwagi et al. [2 as FRs in PP polymers. It was shown that fumed silica and silica gel with a large surface area and a low density can reduce the contact of the polymer with the combustion flame by acting as a thermal barrier layer covering the surface of the molten PP polymer. Thus, they can significantly reduce the total heat release of the PP composite system. However, fused silica, due to its relatively small specific surface area, sinks into the polymer and makes little contribution to thermal insulation and flame retardancy.There are three types of SiOi et al.  explored2 effect on the flame retardancy of PO cable materials [2 and other conventional FRs. The table compares the effect of SiO2 as an adjuvant for flame-retardant system. It is showed that researchers aimed mostly at the EVA polymer. Moreover, with an addition of about 1–5 wt.% SiO2, the LOI and tensile strength of the polymers can be improved noticeably.Recently, many scholars have conducted research about the SiOaterials ,62. TablMMT is a very soft nano-layered silicate consisting of a central sheet of alumina octahedral and two sheets of silica tetrahedral. Accordingly, its structure is shown in However, since the PO matrix is a typical nonpolar polymer material, while the surface of MMT sheet layer is highly hydrophilic and polarized, the compatibility between them is poor and cannot achieve good dispersion, thus seriously affecting the mechanical performance of composites. In order to solve the above problems, one approach is to functionalize PO by introducing polar or polarizable groups into the polymer to convert the nonpolar molecular chains of PO into polar ones . AnotherAccording to the element types and molecular structures, organic phosphorus FRs are divided into three types: phosphorus-containing esters  ,87, phosAs for the phosphorus-containing esters FRs, caged bicyclic phosphate PEPA   and SPDP2 was added in the PP/30% MPHP composites as an adjuvant. 2O7 was formed to cover on the surface, so that MPHP/SiO2 FRs improved the LOI values of PP composites.Polyphosphazene, as a kind of phosphorus–nitrogen adjuvant, has excellent flame retardancy and thermal stability. Due to its special molecular structure and flexible main chain, polyphosphazene can play a positive role in improving the mechanical properties of polymers . Wu et aOrganic silicon has always been in the frontline of co-additives in FRs systems for PO composites because of its characteristics of high efficiency, low smoke evolution, and environmentally friendliness . OrganicThere are two types of organic silicones used as FRs for PO substrates, i.e., additive type and reactive type. The former means that organic silicone is added to the PO substrate by itself or with other FRs without forming chemical bonds with PO matrix. The reactive type mainly refers to the preparation of new flame-retardant polymers by copolymerizing with flame-retardant units and grafting or crosslinking with macromolecules ,106,107.Thus far, researchers have tried a series of halogen-free flame-retardant additives to improve the mechanical performance of PO substrates. Nevertheless, the experimental results rarely showed a good balance between mechanical performance and flame retardancy ,109. To The mode of action of IFR is to add some intumescent additives as flame retardants in the preparation of PO materials that will decompose into an intumescent carbon layer wrapping around the surface of the PO matrix under high-temperature conditions, playing a role in isolating heat, combustible volatile gases, and oxygen, thus blocking the combustion process ,125.In the development process of developing polymers that are flame retardant, phosphorus/nitrogen-based intumescent flame retardants (P-N based IFR), first pioneered by Camino et al.  in 1990,P-N-based IFR is the most abundant and most widely used FR in the IFR systems. The MEL, APP, and PER components are used as the gas source, acid source, and carbon source, respectively . HoweverIn response to the above problems, researchers have conducted a large number of experimental studies, including the surface treatment of P-N-based IFRs, the addition of cooperative FRs, and the synthesis of new IFRs. In the next section, the research progress in these three directions is described in detail.•MicroencapsulationThe different polarities between the IFR and PO matrix leads to the poor compatibility of IFR, which severely reduces the mechanical properties of the PO composites . Up to nZhang et al.  used HBP•Surface modificationSurface modification is another feasible method to get rid of the drawback of incompatibility between IFR and PO. A number of modifiers were applied to promote the interfacial compatibility, such as KH-550, KH-560, and silicone oil ,143, whiTo solve this problem, Lin et al.  used theIn addition, the methods of modification for IFRs have been mentioned in other studies, as given in In addition, researchers found that by adding other FRs, e.g., metal hydroxides, carbon nanomaterials, or silicon-based materials, to replace part of the IFR, the mechanical properties of the PO materials could be improved, while ensuring the flame retardancy. Firstly, these adjuvants make the release of non-combustible gases more stable during the IFR combustion process, thus helping PO polymers to form a dense and stable expanded carbon layer. Secondly, they can also be used as supplements for IFR to enhance the gas phase and condensed phase barrier effects, thereby reducing the risk of combustion in composites and positively impacting the mechanical performance of PO polymers.•Metal-based adjuvantsMetal-based compounds have the function of accelerating the dehydration of PO matrix and IFR to form a compact and stable carbon layer. Moreover, a small amount of metal-based adjuvants added into the PO/IFR cable materials can have a positive effect on the flame-retardancy efficiency.2O3, MnO2, and CeO2) on the carbon-forming mode of action of PP/IFR composites. It was found that these three metal oxides promoted PP/IFR systems to form a more continuous and intensive intumescent carbon layer  as the cooperative agent on the mechanical performance and flame retardancy of PP/IFR system. The cooperative effect could be observed by the enhanced LOI value (from 36% to 36.1%) and UL-94 grade (from V-1 to V-0) of PP/IFR composites with 1.5 wt.% SrCO3. Moreover, the tensile strength of PP composites enhanced from 26.8 to 29.4 MPa compared with the sample without the SrCO3 addition. It was suggested that, on the one hand, SrCO3 could catalyze the chemical reaction between MAPP and PER, thereby promoting the PP polymer to form a stable and dense carbon layer structure. On the other hand, for the phosphate existing in the carbon layer, SrCO3 could generate bridge bonds between them, forming a crosslinked char layer rich in P element, which further improves the stability of the carbon layer. Therefore, the appropriate amount of SrCO3 also contributes to the improvement of mechanical performance of PP composites.Moreover, it was found that transition metal ions, such as Febstrates . Chen etbstrates  studied •Carbon-based adjuvantsThe preparation of polymer/nano-carbon composites has become an effective method to enhance the flame retardancy of materials ,162. TheYang et al.  investig•Silica-based adjuvants2 [https://www.x-mol.com/paperRedirect/1296136865981276160.Recently, silica-based FRs have attracted extensive attention in the research on cooperative flame-retardant PO/IFR composites, such as SiO2 ,175, MMT2 ,177, and2 ,178 httpWen et al.  used OMM•Other adjuvants2Sn2O7  via the hydrothermal method, which can be applied as an adjuvant for PO/IFR composites due to its high-temperature catalytic performance. Moreover, they thoroughly studied the flame retardancy and mechanical performance of the PO/IFR/RES composites. The results showed that RES could enhance the UL-94 classification and LOI value of PO/IFR composites. The LOI of PO/IFR composites increased from 30% to 34%, 33%, and 32%, after adding Nd2Sn2O7, Sm2Sn2O7, and Gd2Sn2O7, respectively. The mode of action of flame retardancy is that the PO/IFR/RES composites can form a more continuous and compact protective carbon layer  all improved the tensile strength of PO/IFR, but they have no obvious effect on the elongation at break.The three types of adjuvants above added to the PO cable materials have achieved significant cooperative improvements in flame retardancy and mechanical properties. For instance, Jia et al.  preparedshown in a, which shown in b. These Moreover, there are various additives used as adjuvants with different IFRs, which are summarized in In addition to the methods of surface treatment and cooperative additives, there is another feasible solution to enhance the distribution of IFR in the PO matrix, i.e., designing a single-component IFR by combining the components of acid, carbon, and gas into a macromolecular structure, the so-called trinity IFR ,199,200.Based on the conventional IFR system of APP/MEL/PER, Yang et al.  synthesiFurthermore, designing new molecules as acid sources or carbon sources for IFR systems also has a certain contribution to improve the performance in flame retardancy and mechanical properties of materials. EG is a layered crystalline carbon-atom-embedded compound. The acid ions between the layers will be released when heated, causing EG to dehydrate and carbonize , thus foWhen Yang et al.  used AHPFurthermore, various studies on the cooperative flame-retardant effect between EG and other additives are listed in In this article, the FR compounds for PO cable sheath materials were reviewed, with emphasis on the cooperative modification of flame retardancy and mechanical properties. For the three typical PO materials, i.e., PP, EVA, and LDPE, 60, CNTs, and graphene), and metal hydroxide . By adding these nanoscale FRs, PO cable sheath materials with improved mechanical properties can be obtained. Furthermore, the use of different kinds of nanoscale FRs or their combination with other P-, N-, or Si-containing FRs is a promising strategy to develop flame-retardant polymer composites with enhanced mechanical properties. In addition, among the FRs used in PO, IFR is attracting more attention due to its good efficiency and environment friendliness. However, there are disadvantages in the traditional IFR system, such as the incompatibility with PO matrix and the high loading, which would make it difficult for the IFR to be homogeneously dispersed in PO matrix, resulting in poor mechanical properties. In response to this problem, the aforementioned three strategies can also be adopted.Due to environmental consideration, more research will be focused on the halogen-free flame-retardant PO cable sheath materials in future. The FR compounds can be divided into inorganic FRs, organic FRs, and IFRs, while the IFRs include both organic and inorganic compounds. Regarding the flame-inhibition strategies, they can be divided into two mechanisms: gas-phase FR mechanism and condensed-phase FR mechanism. However, in many situations, the FRs affect the mechanical performance of the PO matrix due to the poor compatibility between them, especially when a higher loading of FR compounds is utilized. Therefore, when designing a FR formulation for PO cable sheath materials, the comprehensive performance of both flame retardancy and mechanical performance should be considered. To address the compatibility of the PO matrix/flame retardant compounds and enhance the adhesion between them, a series of methods have been adopted, including surface treatment, i.e., microencapsulation and surface modification; ultra-fine treatment; and cooperative combinations of different FRs. Among them, surface treatment techniques are often used in inorganic FR composites, e.g., metal hydroxide, inorganic phosphorus, and inorganic silicon, to overcome the polarity difference between them and the PO matrix, thereby improving the adhesion properties between the two interfaces. At present, the nanoscale FRs obtained by ultra-fine processing are mainly silicon-based materials , carbon-based materials (e.g., CHowever, although some FRs have good performance on flame retardancy, and the obtained PO composites possess acceptable mechanical performance, most of the FRs are still in the stage of laboratory research. New FR materials that can be easily prepared with high efficiency and environmental harmlessness will be the desired ones for flame-retardant PO cable sheath materials in the future.


Friedreich’s ataxia (FA) is an inherited progressive neurodegenerative disease for which there is no proven disease-modifying treatment. Here we perform an open‐label, pilot study of recombinant human granulocyte-colony stimulating factor (G-CSF) administration in seven people with FA (EudraCT: 2017-003084-34); each participant receiving a single course of G-CSF . The primary outcome is peripheral blood mononuclear cell frataxin levels over a 19-day period. The secondary outcomes include safety, haematopoietic stem cell (HSC) mobilisation, antioxidant levels and mitochondrial enzyme activity. The trial meets pre-specified endpoints. We show that administration of G-CSF to people with FA is safe. Mobilisation of HSCs in response to G-CSF is comparable to that of healthy individuals. Notably, sustained increases in cellular frataxin concentrations and raised PGC-1α and Nrf2 expression are detected. Our findings show potential for G-CSF therapy to have a clinical impact in people with FA. Work in a mouse model of Friedreich’s ataxia has shown that administration of the cytokine granulocyte-colony stimulating factor (G-CSF) could have beneficial neuroprotective effects. Here the authors perform a pilot study of Lenograstim (recombinant G-CSF) in patients with Friedreich’s ataxia. In over 95% of cases, the disease is caused by a homozygous GAA.TCC tri-nucleotide repeat expansion mutation within intron 1 of the FXN gene, leading to transcriptional repression of the mitochondrial protein frataxin2. Frataxin has multiple functions including formation of iron-sulphur clusters, mitochondrial homoeostasis and antioxidant protection3. Within the nervous system frataxin deficiency is associated with degeneration of the cerebellum, including the dentate nucleus, dorsal root ganglion neurons, peripheral sensory nerves and the spinal cord1.Friedreich’s ataxia (FA) is a progressive neurodegenerative disease for which there is no proven disease-modifying treatment. It is the commonest hereditary ataxia, affecting at least 1 in every 50,000 people worldwide. Symptoms generally begin in childhood and affected individuals experience progressive accumulation of neurological disability including limb and trunk ataxia, dysarthria, sensory neuronopathy and pyramidal weaknessSince the discovery of the genetic basis for FA there have been significant improvements in our understanding of how frataxin deficiency causes disease. Despite this, progress to identify an effective treatment to limit disease progression and reduce long-term disability has been limited; only symptomatic treatments of modest efficacy are currently available, and none reverse damage or restore neurological function. Disease-modifying therapy for people with FA therefore represents a major unmet clinical need.6. Agents including erythropoietin and interferon-gamma have been tested for their potential to upregulate frataxin with negative outcomes8. Histone deacetylase inhibitors, with the aim of reversing FXN silencing to increase frataxin transcription, have also been examined and early clinical trials of drugs such as nicotinamide have demonstrated evidence of promise in short-term studies9.A variety of different therapeutic strategies for FA have been explored in clinical and preclinical studies. Pharmacological agents have been trialled with the aim of attenuating the downstream effects of frataxin deficiency, namely oxidative stress, iron overload and mitochondrial dysfunction. These agents showed promise in preclinical and early phase studies, but subsequent randomised controlled trials of such drugs including idebenone, deferiprone, or a combination of coenzyme Q10 and vitamin E failed to demonstrate a reduction in disease progression11. Moreover, we have also shown that these agents had neuroprotective effects in FA, increased stem cell mobilisation to sites of pathology and stimulate neural repair10.Continued research and technological advances in regenerative medicine have increased interest in developing neuro-reparative stem cell-based therapies for people with FA. Using a humanised murine model of FA we demonstrated that treatment with the stem cell mobilising cytokines granulocyte-colony stimulating factor (G-CSF) and stem cell factor had pronounced effects on frataxin levels within the nervous system and improved clinical, biochemical and pathological parameters associated with the disease12. Consequently, G-CSF has a well-established safety record in clinical practice and the pharmacokinetics of G-CSF administration have been extensively studied. Together with preclinical evidence supporting a likely beneficial effect and the current lack of effective treatments for FA, the rationale for the clinical trial of disease modification using G-CSF was clearly established.G-CSF is an approved drug widely used medically to treat neutropenia or stimulate the release of haematopoietic stem and progenitor cells from the bone marrow into the peripheral circulation prior to a peripheral blood (PB) stem cell harvestHere, we report the findings of an open-label pilot study of G-CSF administration in seven people with FA. We demonstrate that administration of G-CSF to people with FA is safe and tolerable, leads to effective haematopoietic stem cell (HSC) mobilisation and improves the biochemical profile of molecules relevant to the pathogenesis of FA including increasing frataxin levels. Our findings provide evidence to support an efficacy study of repeated courses of G-CSF in people with FA.n = 2) and mild headache (n = 1), all of which were listed in the trial protocol as known side effects and all resolved immediately after cessation of therapy. There were no serious adverse events. There was no evidence of changes in pulse rate, blood pressure (systolic and diastolic) or body temperature throughout the study (Table P = 0.031)13, but these were not associated with symptoms and resolved spontaneously by day 19. All other parameters were within normal physiological ranges  (Table + cells increased from 7.5 × 106 (SEM ± 1.8) cells per l at baseline to 98.4 × 106 (SEM ± 21.6) following G-CSF administration; CD34+ CD133+ cells rose from 0.9 × 106 (SEM ± 0.4) cells per l to 23.8 × 106 (SEM ± 10.2)   on day 6 showed an appropriately marked rise (approximately 7-fold) in the total circulating number of white blood cells in response to G-CSF administration, corresponding with significantly elevated numbers of circulating neutrophils, lymphocytes, monocytes and eosinophils (1) Table . Mobilis06 SEM ± 1.6 folloPmax values for changes in frataxin expression were positive for all six participants over the duration of the study  and platelets were 2.43 pg per µg of total protein (range 0.54–3.72) and 1.60 pg per µg (range 0.75–2.56) respectively Fig. . ProlongPmax values for changes in aconitase activity were positive in four of the six participants  rose steadily over the study period with maximal 3.70 (day 15) and 3.04 (day 19) mean-fold changes respectively Fig. . For aco16. The expression of PGC-1α, NRF1 and Nrf2 proteins in isolated PB MNCs was measured in response to G-CSF administration , for up to two weeks post treatment, in both PB MNCs and platelets after the 5-day course of G-CSF. Since differences in the time course of frataxin deviations were observed, we also analysed the ‘28. Baseline MNC and/or platelet frataxin concentrations reported within our study were consistent with those reported by others30. Furthermore, extrapolating data from those studies that reported frataxin levels in healthy control patients, frataxin levels in the majority of participants were brought within the predicted range for carriers of the GAA expansion following G-CSF treatment30. Further investigation into the effects of repeated G-CSF dosing on both frataxin expression and clinical measures is therefore warranted.A therapy able to increase the expression of frataxin protein is an attractive treatment approach for FA; residual frataxin protein levels in FA are reduced to 5–35% of healthy individuals, while carriers of the GAA expansion having approximately 50% of normal frataxin expression are asymptomatic3. In response to G-CSF and elevations in MNC frataxin levels, this initial study did not provide convincing evidence of subsequent increases in aconitase or SDH activity, although the mean levels of both remained above baseline for the duration of the study and the maximal change in SDH activity (Pmax) being an increase relative to baseline in five of the six participants. PGC-1α, NRF1 and Nrf2 are key orchestrators of mitochondrial biogenesis and antioxidant responses and their expression/activity is inhibited in cells from individuals with FA, leading to cell injury16. In parallel with increases in frataxin expression, G-CSF therapy-induced rapid and sustained increases in PGC-1α levels followed by a delayed but significant increase in Nrf2. This is consistent with PGC-1α’s role in the regulation of Nrf2 activation31. NRF1, another downstream target of PGC-1α and known regulator of mitochondrial biosynthesis14, was unchanged. Such findings suggest complex cell signalling pathways are involved in response to G-CSF treatment. Moreover, G-CSF-induced activation of PGC-1α and Nrf2 signalling may be another valuable therapeutic mechanism to explore in FA.Oxidative damage and impaired mitochondrial function are both key determinants of cellular damage in FA. Frataxin is crucial for iron-sulphur cluster formation, which is required for the functional activity of the mitochondrial enzymes SDH and aconitasePharmacological interventions to increase frataxin expression and reverse the deleterious effects of frataxin deficiency are attractive therapeutic approaches in FA. We have shown that a course of G-CSF therapy in participants with FA is safe, is associated with effective HSC mobilisation, and leads to significant elevations in frataxin together with improvements in biochemical deficits associated with FA. Nevertheless, the need for future assessment of G-CSF administration on affected tissues, such as the heart and brain, using a range of dose levels and dosing frequencies is required. The long-term safety of sustained G-CSF administration in people with FA is also unknown. The natural rate of disease progression in FA necessitates prolonged trial periods to sufficiently detect changes in clinical measures. This study provides proof-of-principle evidence to support an efficacy study of G-CSF administration in FA, using repeated courses over a longer period.FXN gene). Exclusion criteria included participation in other clinical trials within 30 days of the initial dose of G-CSF; pregnancy, breastfeeding or lactation; current or previous diagnosis of serious medical disorders or illnesses including haematologic disease , splenomegaly, autoimmune disease, pulmonary infiltrate, pulmonary fibrosis or haemoptysis; and clinical abnormalities on baseline blood .We performed an open‐label, single‐site pilot study of Lenograstim  administration in seven people with FA. Participants were recruited via Neurology Clinics at North Bristol NHS Trust . Male and female participants aged over 18 years were eligible to participate if they had a genetically confirmed diagnosis of FA , the East of England – Cambridge East Research Ethics Committee (17/EE/0486) and the University of Bristol, Research Governance Research & Enterprise Development Office. Patients were enrolled in the study between 25 June 2018 to 15 October 2018. Informed written consent was provided by all participants enrolled in the study.Trial participants attended the Bristol Brain Centre, Southmead Hospital  for all clinical assessments and procedures. On day 1, before drug administration, baseline blood tests  were completed, a baseline PB sample was collected for laboratory analysis, and clinical assessment of weight, pulse, blood pressure and temperature taken  were recorded.Whole blood, collected into EDTA Vacutainers™ (BD Bioscience), was centrifugated at 900 × g for 10 min at 4 °C to separate plasma from blood cells. MNCs were isolated from the cell fraction by density gradient centrifugation using Lymphprep™ (StemCell Technologies). Platelets were isolated by centrifugation of plasma (2500 × g for 10 min at 4 °C).32 , anti-CD34-FITC  and anti-CD133-APC  in BD Trucount tubes (BD Biosciences) for 15 min at room temperature. Mature red blood cells were lysed with FACS lysis buffer (BD Biosciences) and samples suspended in phosphate-buffered saline. At least 85,000 events per blood sample were acquired with a BD FACSCanto flow cytometer (BD Biosciences). CD34 and CD133 positive stem cells were quantified using BD FACSDiva v8.0.1 software (BD Biosciences), based on the standardised International Society for Hematotherapy and Graft Engineering gating protocolFrataxin protein, in both PB MNCs and platelets, was measured using the Frataxin Protein Quantity Dipstick Assay Kit (Abcam) according to the manufacturer’s instructions. Total protein concentrations of MNC or platelet lysates were quantified using the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific). In all, 2.5 or 10 µg of total protein from MNCs and platelets, respectively, was loaded per dipstick. Frataxin values were obtained using a standard curve generated using human recombinant frataxin protein (Abcam). Frataxin signal intensity was visualised using a Bio-Rad Universal III Bioplex imager and quantified using ImageJ software . Local background intensity was calculated for each separate dipstick and subtracted from the frataxin signal intensity value. Final frataxin protein values were calculated as pg of frataxin per 1 µg of total protein.The enzyme activities (U) of both aconitase and SDH within PB MNC homogenates were determined using the Aconitase Activity Assay Kit and Succinate Dehydrogenase Activity Colorimetric Assay Kit  according to the manufacturer’s instruction. Absorbance was measured using a FLUOstar OPTIMA plate reader (BMG Labtech) and OPTIMA software . Homogenate total protein concentration was quantified using the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific). Enzyme activity values were calculated as activity per 1 µg of total protein.Quantification of nuclear respiratory factor 1 (NRF1), nuclear factor E2-related factor 2 (Nrf2) and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) was carried out using the Bio-Dot Microfiltration manifold system (Bio-Rad Laboratories). Proteins were extracted from MNCs using Extraction Buffer , transferred to nitrocellulose membrane using gravity filtration, and blocked using 5% bovine serum albumin, before incubation with antibodies to beta-actin , NRF1 , Nrf2  and PGC-1α . Immunoreactivity was detected using a horseradish peroxidase–conjugated goat anti-rabbit IgG  secondary antibody. Protein expression was visualised using ECL Prime Western Blotting Detection reagent  in conjunction with a Bio-Rad Universal III Bioplex imager. Densitometric analysis of protein expression was performed using Image Lab software . Relative expression of NRF1, Nrf2 and PGC-1α was calculated after normalisation to beta-actin levels within the same sample.No sample size calculations were performed. This was a pilot study of seven patients prior to larger phase 2/3 studies. Sample sizes were designed to characterise the outcome measures  and based on the incidence of disease in the population, willingness of patients to consider recruitment, adherence rates and time required for data collection.Pmax). We present these individual data graphically, with each of the measurements taken from an individual being divided by that individual’s baseline level, and presented on a log scale to facilitate the distinction between maximum deviations which are greater than or reduced from the baseline value. The sign test (two-sided) quantified evidence against the null hypothesis that an equal number of participants have Pmax values that are increased or decreased compared to their baseline value, i.e. changes are due to random variation or measurement error rather than a systematic effect of Lenograstim.Analyses were performed using GraphPad Prism (GraphPad Prism version 8.4.1 for macOS (2020), GraphPad Software, USA). All measurements were taken from independent samples/biological replicates provided by each of the study participants at multiple time points. For each measure of protein concentration or enzyme activity, the maximum absolute deviation from the baseline value was determined for each of the six participants (Further information on experimental design is available in the Nature Research Supplementary informationPeer review fileReporting summary