Posthemorrhagic hydrocephalus (PHH) often develops following hemorrhagic events such as intraventricular hemorrhage (IVH) and subarachnoid hemorrhage (SAH). Treatment is limited to surgical diversion of the cerebrospinal fluid (CSF) since no efficient pharmacological therapies are available. This limitation follows from our incomplete knowledge of the molecular mechanisms underlying the ventriculomegaly characteristic of PHH. Here, we aimed to elucidate the molecular coupling between a hemorrhagic event and the subsequent PHH development, and reveal the inflammatory profile of the PHH pathogenesis.CSF obtained from patients with SAH was analyzed for inflammatory markers using the proximity extension assay (PEA) technique. We employed an in vivo rat model of IVH to determine ventricular size, brain water content, intracranial pressure, and CSF secretion rate, as well as for transcriptomic analysis. Ex vivo radio-isotope assays of choroid plexus transport were employed to determine the direct effect of choroidal exposure to blood and inflammatory markers, both with acutely isolated choroid plexus and after prolonged exposure obtained with viable choroid plexus kept in tissue culture conditions.+/K+-ATPase activity was enhanced in choroid plexus isolated from IVH rats, but not directly stimulated by blood components. Inflammatory markers that were elevated in SAH patient CSF acted on immune receptors upregulated in IVH rat choroid plexus and caused Na+/K+/2Cl- cotransporter 1 (NKCC1) hyperactivity in ex vivo experimental conditions.The rat model of IVH demonstrated PHH and associated CSF hypersecretion. The NaCSF hypersecretion may contribute to PHH development, likely due to hyperactivity of choroid plexus transporters. The hemorrhage-induced inflammation detected in CSF and in the choroid plexus tissue may represent the underlying pathology. Therapeutic targeting of such pathways may be employed in future treatment strategies towards PHH patients.The online version contains supplementary material available at 10.1186/s12987-022-00360-w. Posthemorrhagic hydrocephalus (PHH) is a condition of progressive ventricular enlargement and elevated intracranial pressure (ICP) that develops following hemorrhagic events such as intraventricular hemorrhage (IVH) and subarachnoid hemorrhage (SAH). The condition is associated with increased morbidity and mortality . PHH can+/K+/2Cl− cotransporter NKCC1 [Choroid plexus CSF hypersecretion was recently demonstrated to contribute to PHH development in an animal model of IVH . CSF hyper NKCC1 , a key cer NKCC1 . It can er NKCC1  and/or ber NKCC1 . PHH couer NKCC1 , 14. Sucer NKCC1 , 18. Hener NKCC1 , 14, 16.Animal experiments performed at University of Copenhagen conformed to the legislations for animal protection and care in the European Community Council Directive (2010/63/EU) and followed all ethical regulations under animal permission no. 2018-15-0201-01595 authorized by the Danish Animal Inspectorate. Animal experiments performed at University of Michigan were approved by the University of Michigan Committee on the Use and Care of Animals and followed the Guide for The Care and Use of Laboratory Animals . Adult male Sprague Dawley rats at 9 ± 1 weeks of age  were used for all experiments. The rats were housed with a 12:12 light cycle and had free access to food and water.−1 air / 0.1 l min−1 O2) to induce anesthesia in an induction chamber and 2–2.5% to maintain anesthesia through a face mask.For the majority of the experiments, rats were anesthetized with an intraperitoneal injection of xylazine and ketamine . Pentobarbital  was applied for the IVH surgeries (described below) in which animals were later subjected to magnetic resonance imaging (MRI). Isoflurane  was applied for the MRI, as well as for the IVH surgeries in which CSF was later collected for analysis and choroid plexus collected for RNA sequencing. Isoflurane was employed using 5% (mixed with 1.8 l minRats were anesthetized and core body temperature was maintained at 37 °C by a homeothermic monitoring system (Harvard Apparatus or Yellow Springs Instrument). The femoral artery was catheterized and approximately 300 µl of arterial blood was collected. Rats were positioned in a stereotaxic frame (Kopf Instruments or Harvard Apparatus), the skull exposed with a midline incision, and a cranial burr hole drilled above the right lateral ventricle . 200 µl of autologous blood was injected stereotactically into the right lateral ventricle  with a 27-gauge needle to induce IVH . The bloAnesthetized rats underwent MRI in a 9.4-Tesla MR scanner 24 h post IVH. The MRI protocol comprised a T2 fast spin-echo and T2* gradient echo sequence with the following settings: repetition time = 4000 ms and effective echo time = 60 ms for T2 weighted imaging; repetition time = 250 ms and effective echo time = 5 ms for T2* weighted imaging. Other parameters included field of view = 35 × 35 mm, matrix = 256 × 128, and slice thickness = 0.5 mm for both T2 and T2* weighted imaging. Twenty-five coronal slices were acquired to cover the entire ventricular system. MRI analysis was performed in Image J (v 1.52a) . LateralThe brains were quickly removed from anesthetized rats following decapitation, and the wet weight was immediately determined on a pre-weighed porcelain evaporation beaker. Brains were afterwards homogenized with a steel pestle, placed in a pre-heated oven at 100 °C, and left to dry for 3 days. The dry brain was weighed, and the brain water content was determined in ml/g dry weight using the equation: (wet weight − dry weight)/dry weight.2, 1.3 MgSO4, 1 NaH2PO4, 10 glucose, 25 NaHCO3, pH adjusted with 95% O2/5% CO2). Ex vivo experiments (radio-isotope flux assays) were conducted in HEPES-containing aCSF  120 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgSO4, 1 NaH2PO4, 10 glucose, 17 Na-HEPES, adjusted to pH 7.4 with NaOH) as continuous gas-equilibration of the HCO3− buffered aCSF is technically challenging with small quantities of isotope-containing test solutions. Initial test experiments revealed similar 86Rb+ efflux rates whether the experiments were conducted in regular gas-equilibrated aCSF or HEPES-aCSF . The agents were either dissolved directly into the solution on the day of the experiment or divided into working aliquots and stored at − 20 °C to avoid repeated freeze-thaw cycles. All control solutions included the appropriate vehicle. The fluorescent dye dextran  was dissolved directly in the heated and gas-equilibrated aCSF prior to use. The incubation time of the applied pharmacological agents varied from acute exposure  to long term exposure (16 h).In vivo experiments were conducted with heated and gas-equilibrated artificial CSF  127 NaCl, 2.5 KCl, 2.5 CaCl® Plus, Starr Life Sciences) to ensure physiologically relevant blood gas levels. Anesthetized and ventilated rats were positioned in a stereotactic frame (Harvard Apparatus) and the skull exposed through a midline incision. A 4.5 mm brain infusion cannula  was inserted into the right lateral ventricle through the cranial burr hole made during the PHH procedure . A cranial window (approximately 3.6 mm in diameter) was drilled on the contralateral side of the skull and an epidural probe  was lowered and secured with dental resin cement . The ICP cannula was pre-filled with HEPES-aCSF and connected to a pressure transducer (APT300) and transducer amplifier module TAM-A (both from Hugo Sachs Elektronik). The pressure signal was recorded with a 1 kHz sampling rate using BDAS Basic Data Acquisition Software . To assure a continuous fluid column between the dura and the epidural probe, approximately 10 µl of HEPES-aCSF was injected through the epidural probe after which jugular compression was applied to confirm proper ICP recording. After observing a stable ICP signal, the measurement was continued for 15 min and the average ICP was determined. The recording and analysis of ICP signals were performed in a blinded manner. The ventriculo-cisternal perfusion procedure [p = CSF production rate (µl/min), ri = infusion rate (µl/min), Ci = fluorescence of inflow solution (a.u.), Co = fluorescence of outflow solution (a.u.).Rats were anesthetized and core body temperature was maintained at 37 °C by a homeothermic monitoring system (Harvard Apparatus). Surgical tracheotomy was performed to obtain mechanical ventilation, which was controlled with the VentElite system (Harvard Apparatus). Ventilation settings were optimized for each rat using a capnograph  and a pulse oximeter (MouseOxrocedure –23 was irocedure : \docume® Ultra™ RNA Library Prep Kit (New England Biolabs) prior to RNA sequencing (RNAseq)  on an Illumina NovaSeq 6000 (Illumina). The 150 base paired-end reads were mapped to reference genome (Rattus norvegicus Rnor_6.0) using Spliced Transcripts Alignment to a Reference (STAR) RNA-seq aligner (v 2.7.2a) [https://github.com/Sorennorge/posthemorrhagic_hydrocephalus. We defined differentially expressed genes as ≥ 20% different and with a TPM difference of ≥ 1 TPM. The obtained data are divided into those with TPM ≥ 0.5 for both conditions (control and PHH) and those with TPM < 0.5 in one of the two conditions with a subdivision, when required, illustrating those without detection (0 TPM) in one of the two conditions  obtained from rats undergoing the IVH procedure or saline-injected sham operated animals were pooled according to experimental condition, (n = 4 of each) and stored in RNAlater  at − 80 °C. RNA extraction and library preparation were performed by Novogene Company Limited, UK with NEB Next 2.7.2a) . The map 2.7.2a) . Gene in 2.7.2a) –28, from 2.7.2a) –31 were  supplemented with penicillin-streptomycin , rat epidermal growth factor , and fetal bovine serum , and incubated at 37 °C, 5% CO2, for 16 or 24 h prior to initiation of subsequent experiments.Isolated rat brains were immersed in ice-cold HEPES-aCSF for 10 min before isolation of the lateral choroid plexus. In a subset of experiments, the acutely isolated lateral choroid plexus was placed in Dulbecco’s Modified Eagle’s Medium 86Rb+)  and 3H-mannitol . 86Rb+ acts as a congener for K+ and therefore readily enters the cells, whereas 3H-mannitol remains on the outside and serves as an extracellular marker [2 (to prevent efflux of intracellular 86Rb+ during the washing procedure), followed by transfer into scintillation vials containing 100 µl Solvable . For efflux, the choroid plexus was swiftly rinsed in 37 °C isotope-free HEPES-aCSF followed by transfer into new wells containing 37 °C isotope-free HEPES-aCSF at 5–20 s intervals. For every time point, 200 µl of surrounding HEPES-aCSF was subsequently collected and placed into scintillation vials. At the end of the experiment, the choroid plexus was placed into a scintillation vial containing 200 µl Solvable. For both influx and efflux, the choroid plexus was dissolved completely before the isotope content was determined in 2 ml Ultima Gold™ XR scintillation liquid  using the Tri-Carb 2900TR Liquid Scintillation Analyzer (Packard). The 86Rb+ activity was corrected for extracellular background using 3H-mannitol [86Rb+ activity at each time point (AT) normalized to the initial 86Rb+ activity (A0) as a function of time. The slope from linear regression analysis was used to determine the 86Rb+ efflux rate constant (min−1) [The isolated lateral choroid plexus was placed in 37 °C HEPES-aCSF for a 10-min recovery period followed by 2 min (influx) or 5–10 min (efflux) of incubation in an isotope solution containing: rubidium ( (min−1) , 32.Calcein acetoxymethyl  staining was employed to verify choroid plexus epithelial cell viability . Viable Choroid plexus, acutely isolated or after up to 24 h of tissue culture, was fixed in 2% glutaraldehyde in phosphate buffer, pH 7.2 before standard Epon embedding for conventional transmission electron microscopy (TEM). TEM sections (60 nm) were examined in a CM 100 microscope and images acquired with an Olympus Veleta camera at a resolution of 2048 × 2048. Thicker sections were stained with toluidine blue for light microscopical examination.g for 5 min at 4 °C prior to protein concentration determination with the DC Protein Assay (Bio-Rad). 10–20 µg protein was loaded on precast SDS-PAGE gels  and transferred using immobilon-FL membranes (Merck Millipore). Primary and secondary antibodies were diluted 1:1 in Odyssey blocking buffer (LI-COR): PBS-T. Primary antibodies: anti-GAPDH; AB2302 , anti-NKCC1; S022D , anti-Na+/K+-ATPase α1; a6F . Secondary antibodies, all 1:10,000; IRDye 680RD donkey anti-chicken , IRDye 800CW goat anti-mouse , rabbit anti-sheep . Images were obtained by the Odyssey CLx imaging system and analyzed by Image Studio ver. 5.2 (LI-COR).Isolated lateral choroid plexus was lysed in RIPA buffer  supplemented with the protease inhibitors pefabloc  and leupeptin , sonicated , and centrifuged at 10,000 × g for 10 min at 4 °C prior to aliquoting and storage at − 80 °C until use to avoid repeated freeze thaw cycles.Anesthetized rats were positioned in a stereotactic frame (Harvard Apparatus) and the cisterna magna quickly (within 10 min) punctured in the same manner as described for ventriculo-cisternal perfusion (see above). The CSF was collected in polypropylene tubes  and immediately centrifuged at 2000 × CSF samples were collected from 12 patients  with acute SAH admitted and treated for the condition at the Department of Neurosurgery at Rigshospitalet, Copenhagen, Denmark. CSF samples were obtained within 24 h of ictus (8/12) or as soon as possible hereafter (4/12) through an external ventricular drain (EVD) inserted on clinical indications. Patients were selected for the present study if they were subsequently diagnosed with PHH and received a permanent ventriculo-peritoneal shunt because of continued need for CSF diversion. As control subjects, 13 patients undergoing preventive surgery for unruptured aneurysms (vascular clipping) were enrolled , and CSF was collected from the basal cisterns during surgery prior to clipping of the aneurysm. Written informed consent was obtained from all patients or next of kin depending on the capacity of the patients and the study was approved by the Ethics committee of the Capital Region of Denmark (H-19001474 and H-17011472/69197) and the Danish Data Protection Agency (P-2019-773).https://www.olink.com/products-services/target/inflammation/) by BioXpedia A/S  using the proximity extension assay (PEA) technique (Olink Bioscience). The PEA technique enables simultaneous detection of 92 inflammatory markers in a CSF volume of only 1 µl, thus providing a high throughput while maintaining excellent sensitivity and specificity [2 scale. The limit of detection (LOD) was calculated for each inflammatory marker as the background signal plus three times the standard deviation of the inflammatory marker. Inflammatory markers were excluded from further analysis if  > 35% of the samples were below the LOD and no preferential presence in any study group was detected. This criterion led to the exclusion of 34 inflammatory markers for the human CSF samples and 78 inflammatory markers for the rat CSF samples. The remaining inflammatory markers  were included in the statistical analysis. For normally distributed data, an unpaired two-tailed t-test was conducted and Welch’s correction was applied if variances were unequal. For non-normally distributed data, a Mann-Whitney U test was conducted. P values were corrected for multiple comparisons using the Bonferroni method [Rat and human CSF samples were analyzed for presence of 92 different inflammatory markers , which was absent in control rats undergone sham surgery on the femoral artery and intraventricular delivery of saline  compared to the control rats  into the right lateral ventricle of anesthetized rats. Ventricular blood deposits were demonstrated in these rats 24 h later by T2* weighted MR scans  displayed ~ 30% upregulation. The accessory β3 subunit of the Na+/K+-ATPase was similarly augmented as was the basolaterally-located Na+-driven chloride bicarbonate exchanger, NCBE. Hence, various choroidal transport proteins implicated in CSF secretion demonstrate an upregulation at the transcriptional level in the setting of PHH in rats.As CSF hypersecretion could arise from IVH-mediated upregulation of choroidal transport mechanisms, we determined the transcriptional abundance of select membrane transport proteins involved in CSF secretion. RNAseq on choroid plexus obtained from PHH rats and control rats 24 h post IVH revealed transcription of 13,884 genes in the choroid plexus. Of the transcripts detected at ≥ 0.5 TPM, 5,944 were considered differentially transcribed between the two experimental groups, with the vast majority (96%) being upregulated in PHH rats compared to their control counter-parts  were similar whether obtained in choroid plexus from PHH rats or their control counterparts  was significantly increased in PHH rats compared to control rats  decreased the 86Rb+ efflux rate at time points t = 0 h and t = 16 h  and the bumetanide-sensitive 86Rb+ efflux (the NKCC1 activity) was thus similar (P = 0.49), Fig. P = 0.11) and most NKCC1-mediated 86Rb+ efflux was thus abolished upon lengthy culturing. Ouabain-sensitive 86Rb+ influx (the Na+/K+-ATPase activity) persisted after 16 h tissue culturing of the choroid plexus . These data suggest that the choroid plexus is viable with persistent transport mechanisms after 16 h in culture medium and can be employed to elucidate effects on prolonged exposure to blood or its break-down product hemin.PHH usually develops over hours to days and blood exposure may thus exert its effect on the choroidal tissue on a similar time scale. We therefore established a culture regime for acutely isolated intact choroid plexus with which the effects of prolonged exposure to blood or its break-down product hemin could be determined. The choroid plexus is a monolayer of epithelial cells, which are all in contact with the gas-equilibrated culture medium as in conventional cell culture. The viability of the cultured choroid plexus epithelium was confirmed with a calcein-AM survival assay , which drol Fig. d. To revrol Fig. f, i. Aft86Rb+ efflux was not significantly affected by 16 h exposure to blood  and control rats (n = 17) collected 24 h post IVH using the proximity extension assay (PEA) technique, which relies on dual antibody-based recognition for detection of 92 different inflammatory markers . However and contTo determine whether PHH, possibly via its associated inflammatory response, altered the rat choroid plexus transcriptome, and thus potentially its CSF secretory properties, we searched the 5944 differentially transcribed genes  that were both elevated in the SAH patient CSF and also target the inflammatory receptors that were upregulated in choroid plexus from IVH rats Fig. b. The miWe here demonstrate that an animal model of IVH presents with the ventriculomegaly and excessive brain water accumulation characteristic of the PHH observed in patients with cerebral hemorrhagic events . We revePHH arises following hemorrhagic events of different kinds, e.g. SAH and IVH . The con+/K+-ATPase [+/K+-ATPase activity was elevated in choroid plexus acutely excised from PHH rats. Such difference could be explained by the different experimental approaches, i.e., in vivo determination of NKCC1-mediated CSF secretion [+/K+-ATPase activity may mask the effect of PHH-induced increased NKCC1 activity by lowering intracellular Na+, reducing the electrochemical gradient for efflux via the co-transporter. Elevated Na+/K+-ATPase activity in the choroid plexus from IVH rats could arise by a similar phosphorylation-based increase in activity [+/K+-ATPase, which is highly expressed in the choroid plexus [+/K+-ATPase α2 isoform, which is expressed at substantially lower levels than the α1 isoform [ATP1A2, Additional file +/K+-ATPase isoform has previously been implicated in LPS/TLR4/NF-κB-mediated immune responses [CSF secretion by the choroid plexus occurs, in part, by the action of NKCC1 and the Na+-ATPase , 48–51, +-ATPase . These w+-ATPase . In our ecretion  versus iactivity , by the d plexus , or by eesponses , 56, a fesponses . The IVHesponses .+/K+-ATPase when applied to choroid plexus ex vivo. Choroidal hypersecretion could, instead, arise as a function of the inflammatory response associated with the hemorrhagic event. Morphological examination of choroid plexus revealed that these retained their structural integrity following the hemorrhagic event but displayed transcriptional upregulation of a wealth of inflammatory markers and immune receptors, including different subunits of the NF-κB transcription factor complex, which has previously been implicated in IVH-mediated CSF hypersecretion [We demonstrated that the IVH-mediated CSF hypersecretion mostly likely does not arise as a direct function of choroidal exposure to blood, or its breakdown product hemin [an established TLR4 agonist , as thesecretion . Our finWe here performed an unbiased screening of 92 different inflammatory markers in ventricular samples of CSF obtained during the diagnostic workup of a set of SAH patients with later development of PHH. These CSF samples were compared to CSF collected from healthy control individuals undergoing prophylactic neurosurgical removal of unruptured aneurysms. As it is considered unethical to access the ventricular compartment of healthy individuals without a clinical indication, the CSF from this control group was collected from the basal cisterns during the neurosurgical procedure. The different CSF collection sites in the two test groups is ethically unavoidable but must be considered a limitation of the present study. The CSF obtained from the SAH patients presented both up- and downregulation of various inflammatory markers. The majority of the upregulated inflammatory markers were chemokines  or interleukins , which promote and regulate immune responses in a pleiotropic manner, depending on their target , 60. TheDirect exposure of isolated rat choroid plexus to the rat version of the inflammatory markers observed in SAH patient CSF, the receptors of which were also upregulated at the transcriptional level in IVH rats, led to NKCC1 hyperactivity ex vivo. It should be noted that these results were obtained with only a subset of the 10 elevated inflammatory markers acting on only a subset of the 92 immune receptors with increased transcript abundance in the rat choroid plexus. We selected those that were upregulated in both CSF and choroid plexus to obtain a proof of concept. The remaining elevated inflammatory markers could well promote a similar NKCC1 activation even without transcriptional upregulation of their receptors and vice versa, alone or in combination. It thus appears that a hemorrhage-induced inflammatory response enables the transporter hyperactivation leading to the CSF hypersecretion observed in connection with the hemorrhagic event . Its comSecretion and circulation of CSF in quadrupeds may differ from that of human. Therefore, limitations to the study include the use of rodents for the invasive in vivo quantification of CSF secretion rates and for all ex vivo determinations of choroid plexus transcript abundance and transport activity. These processes cannot be studied in the humans. An additional limitation is that the human patient group consists of aged individuals , whereas the basic mechanisms of IVH-induced PHH formation were performed on young adult rats (9 weeks). We cannot rule out that age influences the molecular mechanisms underlying IVH-induced PHH and that the contribution of drainage blocking versus CSF hypersecretion may vary over time after the hemorrhagic event and with the type event .+/K+-ATPase and NKCC1 hyperactivation. The underlying molecular coupling remains unresolved but is likely to be associated with the inflammatory response observed with a hemorrhagic event in patient CSF and rat choroid plexus. Hemorrhage-mediated hypersecretion may serve to swiftly rid the ventricular system of the blood introduced with the hemorrhage, but in some cases promote the ventriculomegaly and elevated ICP observed in this patient group. Future revelation of the molecular etiology underlying PHH-mediated modulation of CSF secretion and the associated regulatory immune pathways may provide rational therapeutic targets towards pharmacological treatment of this condition.In conclusion, a component of PHH development may be linked to CSF hypersecretion, due, in part, to choroid plexus NaAdditionalfile 1: FigureS1. Fluxassay, Western blot.Additionalfile 2: FigureS2. Flowdiagram for the CSF analysis. Additionalfile 3: TableS1. RNAseq_PHH_rat_all genes.Additionalfile 4: TableS2. Inflammatory markers for statistical analysis in rat CSF samples.Additionalfile 5: TableS3. Inflammatory markers for statistical analysis in human CSF samples.Additionalfile 6: TableS4. Elevated inflammatory markers in CSF from PHH patients versus healthy control subjects.Additionalfile 7: TableS5. Decreased inflammatory markers in CSF from PHH patients versus healthy control subjects.Additionalfile 8: TableS6. RNAseq_rats_PHH_Immune_Receptors.Additionalfile 9: TableS7. RNAseq_rats PHH_Immune_Response.


The associations between CSF ferritin (indicating iron burden) and sTrem2, as well as AD pathology, which is reflected by Aβ42, t-tau, and p-tau in CSF, were explored. CSF ferritin was significantly associated with sTrem2 among all participants , HC , and AD continuum , respectively. However, ferritin predicted the accelerated sTrem2 level in those with high ferritin . In conclusion, CSF ferritin serves as a potential biomarker of Trem2-indicated microglia function.Brain iron accumulation, which is indicated in the cerebrospinal fluid (CSF) ferritin, is associated with the development of Alzheimer's Disease (AD). Studies have indicated that iron deposition might participate in Alzheimer's pathology through the induction of microglial activation. A soluble triggering receptor expressed on myeloid cells 2 (sTrem2) in CSF is increasingly recognized as a reliable indicator for microglia activity in the brain and participates in the development of neuroinflammation. However, the association between CSF ferritin and sTrem2 under the AD continuum has not been well-established. We enrolled individuals from the Alzheimer's Disease Neuroimaging Initiative (ADNI) database. Participants were classified into healthy controls (HC,  Iron homeostasis is pivotal for the operation of the central nervous system (CNS) . The metvia their intrinsic abilities of secretion, chemotaxis, and clearance. Those activated cells would turn into an inflammatory phenotype in AD, exhibiting the typical morphological changes and producing inflammatory factors  mode was used in this study to evaluate the natural progression process of Alzheimer's dementia, which offers a comprehensive and reasonable way of taking both pathological and clinical presentations into account . We usedwww.adni-info.org. The participants included in the current study were available for CSF ferritin, CSF sTrem2, ApoE4 presence, MMSE, and demographic information, including age, gender, and education. Alzheimer's biomarkers, indicated by CSF Aβ42, total-tau (t-tau) and phospho-tau (p-tau), and CDR were acquired to facilitate the clinical-A/T/N classification.Data used in the current study were obtained from the ADNI database (adni.loni.usc.edu). Detailed information of ADNI enrollment have been described previously . ADNI waA/T/N scheme is a biomarker-based profile, which aims to facilitate the diagnosis of AD. According to the 2018 NIA-AA “research framework” for the diagnosis of AD , the A/Tn =24), and symptomatic individuals without A+ (n = 21) or TN+ (n = 43), were excluded from the following analysis. Details of the participants are shown in Then, a combination of the A/T/N biomarker profile and the clinical status (CDR) was used to classify the included participants . Clinicahttps://ida.loni.usc.edu).CSF biomarker profiles were established according to the protocols used in ADNI. CSF sTrem2 was measured based on the MSD platform using the ELISA assay established by the Haass' group (available in MSD-sTREM2CORRECTED.csv in the ADNI database). CSF was measured by the electrochemiluminescence immunoassays Elecsys Aβ42, t-tau, and p-tau on a fully automated Elecsys cobas e 601 instrument (available in UPENNBIOMK9.csv in the ADNI database). Details of the CSF biomarkers assessments can be found online in the ADNI database . Baseline characteristics between HC and AD continuum were compared. All analyses were done on all individuals and separate population using analyses of variance (ANOVA) for continuous data and chi-square tests for categorical measures. CSF Aβ42, t-tau, p-tau, ferritin, and sTrem2 were log-transformed to reduce skewness. We analyzed the associations of CSF ferritin with sTrem2 or AD biomarkers  using linear regression models, with age, gender, and ApoE4 presence as covariates. Moreover, the models were also established to assess the associations of CSF sTrem2 with fluid biomarkers. Subgroup analyses were done on HC, AD continuum, and ferritin positive/negative (based on median level of ferritin: 6.4 ng/ml) populations. Moreover, results were adjusted for multiple testing by calculating false discovery rate (FDR)-corrected n = 105) to limit the number of comparison groups (P < 0.05).The study enrolled 151 participants from the ADNI database. Using an established ATN-CDR criteria, they were classified into 46 HC, 20 PreAD, and 85 AD patients. PreAD and AD participants were merged into an AD continuum group  , and patients classified into AD continuum , but not in participants with low ferritin  . The samP = 0.605, FDR = 0.649), t-tau  and p-tau  in any participants  and p-tau  levels in all individuals. The significant results were also found in the AD continuum , but not in healthy participants (P > 0.05). Moreover, the status of ferritin burden did not alter the trends of their associations.We also investigated the predictive values of CSF sTrem2 with AD pathology . It indiThe study substantiates the interplay mode between iron and microglia in AD, by demonstrating the positive associations of CSF ferritin with increasing sTrem2 under an evaluation trajectory using an ATN framework and CDR-defined clinical status. Moreover, we investigated the associations of CSF ferritin with neurodegenerative biomarkers, including Aβ42, t-tau, and p-tau across the AD continuum.sTrem2 in CSF is used to indicate the activity of microglia in the brain , 24. TheStudies have also indicated that the content of iron increases in the brain of aging populations and patients with AD . We trieOf note, an ATN-CDR criterion was used in this study to evaluate the natural progression process of Alzheimer's dementia, which has been proven to offer a comprehensive and reasonable way to take both pathological and clinical presentations into account . When weAlthough the study added to our understanding of the potential role of CSF ferritin in the context of AD in a clinical setting, there were some limitations in the current project. In this study, brain iron burden was manifested as the ferritin in CSF. Ferritin is the main source of iron in the brain. CSF ferritin should serve as an indirect marker to demonstrate the iron burden of whole brains. Second, the results came from a cross-sectional setting. Further investigation under a longitudinal scope would help the translational application of the findings. Third, more neurodegeneration markers, like neurofilament light chain may be considered for future studies, to ensure the overall analysis of the effects of ferritin on neurodegenerative changes. Moreover, ATN mode is truly a promising evaluation system to understand the progression of AD. Its combination with clinical rating system may need more supporting information.In conclusion, we demonstrate that CSF ferritin associates with sTrem2, even in the absence of AD pathology. These findings suggest that CSF ferritin could serve as a surrogate biomarker of Trem2-indicated microglia function.https://adni.loni.usc.edu/data-samples/access-data/.The data analyzed in this study was obtained from the Alzheimer's Disease Neuroimaging Initiative (ADNI), the following licenses/restrictions apply: The application process includes acceptance of the Data Use Agreement and submission of an online application form. The application must include the investigator's institutional affiliation and the proposed uses of the ADNI data. ADNI data may not be used for commercial products or redistributed in any way. Requests to access these datasets should be directed to ADNI, The studies involving human participants were reviewed and approved by Alzheimer's Disease Neuroimaging Initiative (ADNI) and ADNI Data Sharing and Publications Committee. The patients/participants provided their written informed consent to participate in this study.adni.loni.usc.edu), which is easily available for download from the Laboratory of Neuroimaging (LONI) website to the research public. As such, the investigators within the ADNI contributed to the design and implementation of ADNI and/or provided data but did not participate in analysis or writing of this report. A complete listing of ADNI investigators can be found at: http://adni.loni.usc.edu/wp-content/uploads/how-to-apply/ADNI-Acknowledgement-List.pdf.Data used in this article were obtained from the ADNI database , Guangzhou Municipal Key Discipline in Medicine (2021–2023), National Natural Science Foundation of China (Grant No. 821715333), Foundation of Guangdong Province (2022A1515011623), and Medical Scientific Technology Research Foundation of Guangdong Province (Grant No. A2020446).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.


We applied multiple co-inertia analysis (MCIA), an unsupervised and thereby unbiased, multivariate method for simultaneous data integration and found that the top latent variable classifies RRMS status with an Area Under the Receiver Operating Characteristics (AUROC) score of 0.82. Variable selection based on Lasso regression reduced features to 44, derived from the small RNAs from plasma (20), CSF cells (8) and cell-free CSF (16), with a marginal reduction in AUROC to 0.79. Samples from SPMS patients (n = 6) were subsequently projected on the latent space and differed significantly from RRMS and controls. On contrary, we found no differences between relapse and remission or between inflammatory and non-inflammatory disease controls, suggesting that the latent variable is not prone to inflammatory signals alone, but could be MS-specific. Hence, we here showcase that integration of small RNAs from plasma and CSF can be utilized to distinguish RRMS from SPMS and neurological disease controls.Multiple Sclerosis (MS) is an autoimmune, neurological disease, commonly presenting with a relapsing-remitting form, that later converts to a secondary progressive stage, referred to as RRMS and SPMS, respectively. Early treatment slows disease progression, hence, accurate and early diagnosis is crucial. Recent advances in large-scale data processing and analysis have progressed molecular biomarker development. Here, we focus on small RNA data derived from cell-free cerebrospinal fluid (CSF), cerebrospinal fluid cells, plasma and peripheral blood mononuclear cells as well as CSF cell methylome data, from people with RRMS ( Multiple Sclerosis (MS) is a complex autoimmune and neurodegenerative disease, which can manifest with clinical symptoms such as impaired vision, fatigue, sensory disturbance, spasticity, pain, and depression among others . Due to Molecular biomarkers, such as immunoglobulin G (IgG) index, oligoclonal bands and neuro-filament light chain in the cerebrospinal fluid (CSF) are becoming more widely used for supporting MS diagnosis together with patient medical history, clinical examination and magnetic resonance imaging (MRI) emphasizing the importance of moving towards multidisciplinary approaches in disease management . Listed Previous studies have predominately considered tissue-specific, single omics data, in search of biomarkers. However, recent advances in omics technologies have invited multi-omics and multi-tissue biomarkers to the scene, which allows diagnostic biomarkers and treatment response evaluation to be multi-layered . BioinfoIn this study, we applied a multi-omics, multi-source integration approach to showcase MS biomarker discovery. We profiled small RNAs in CSF (cell-free and cells) and blood (plasma and mononuclear cells) with Small-seq methodology . FurtherRRMS = 20, nSPMS = 5 (+1), nCIS = 1, nRIS = 1) and neurological controls  as previously described (g for 15 min at room temperature (RT), followed by plasma phase aspiration. CSF was centrifuged immediately after collection at 440 g for 10 min at RT to separate cells from supernatants. All samples were stored at −80°C until further processing.Blood and CSF were collected from people with MS  protocol was conducted as previously described  on lysathttp://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Quality (-q 20) and adapter (-a AGATCGGAAGAGC) trimming were performed using Cutadapt 1.8.1 (Reads were trimmed 9bp from their 5′end (--clip_rl 9) using Trim Galore version 0.4.1  = median (xi - median(x))/median(x)) lower than one and standard deviation of 0 across samples were filtered.The BSmooth function from bsseq v.1.20.0  was applk refers to expression matrix X from dataset K. Xpk is the mean of the column p and σpk is the standard deviation of column p. The resulting standardized datasets were then combined followed by singular value decomposition (SVD) to calculate the first eigenvectors:kT refers to the eigenvectors for variables from dataset k. The collection of all UΣ  is referred to as global latent scores and is referred to as Tglobal. The partial contributions for each subtype were divided by the contributions of the complete dataset to make fractions and percentages. For integration, we performed multi co-inertia analysis (MCIA)  by applyXnew is the new unseen standardized dataset  and VT is the SVD weights. The variance contribution for each omics was calculated as:For projection and estimation eigenvalues for subtypes, we used only the corresponding eigenvectors to estimate latent scores and variance contribution. More specifically, the projections were calculated by:1.Apply k-means clustering with k = 22.Compute the fractions RRMS (RRRMS) and Controls (Rcontrol) detected in cluster one and cluster two3.Determine the true positive cluster by comparing RRRMS and Rcontrol such that cluster one is true positive if RRRMS > Rcontrol otherwise cluster two is the true positive4.Calculate true positive and false positive fractions5.Relocate a sample xi from cluster one to cluster two such that the change in the k-means objective function is minimum6.Calculate true positive and false positive fractions7.Repeat steps five to six until cluster one becomes an empty cluster8.Reinstate the k-means clustering results9.Relocate a sample from cluster two to cluster one such that the change in the k-means objective function is minimum10.Calculate true positive and false positive fractions11.Repeat steps 9–10 until cluster two becomes an empty clusterClustering was performed using k-means with k = 2. We calculated the accuracy using the split-join distance  and the λ. We used the global scores from MCIA as the dependent and the features as the independent variable. The features with shrunk ß = 0 were removed. If no variable from a dataset was left, the entire dataset was removed before performing MCIA on the reduced dataset. The statistical inference was performed using Mann-Whitney test. p-values lower than 0.05 were considered statistically significant.Variable selection was performed using Lasso regression  , RRMS during remission (n = 9), SPMS , as well as inflammatory  and non-inflammatory neurological disease controls . Furthermore, samples from two individuals classified as RIS and CIS at the time of collection, were also included  were collected from RRMS in relapse  based on L1 was evaluated with line, box- and density plots (p < 0.001) , which is an unsupervised and thereby unbiased method . MCIA isty plots . Wilcoxo< 0.001) . Partial< 0.001) .p < 0.001) based on Wilcoxon signed-rank test   . Selecte< 0.001) , CSF (We< 0.001)  and plas< 0.001)   . Hence, In summary, Lasso regression reduced the number of input features to 44, while consistently allowing for classification of RRMS. Hence, we here demonstrate that data from multi-cellular and fluid sources can be reduced to few features, which is important for future biomarker potential.Ranking of individual MCIA loadings from the 44 selected features revealed that among top 15, 12 were tsRNAS derived from MINTmap profiling with the majority derived from the CNS compartment and down-regulated in RRMS compared to controls . HoweverIn summary, a mix of transcript biotypes and sources contribute to the latent variable of the reduced model with major contributions coming from misc- and miRNAs from CSF cells and CSF and a 3′tRF from plasma.In the current study, we utilized an unsupervised approach, based on simultaneous integration of omics data from fluids and cells from the same individuals to distinguish MS from controls. Samples are derived from blood or CSF, which are both of biological relevance to MS due to the immunological component of the disease and proximity of CSF to the target organ. Furthermore, blood and CSF are routinely collected in MS diagnostic evaluation emphasizing their biomarker potential. We focused on small RNAs and DNA methylation and found that small RNAs from cell-free CSF, CSF cells and plasma played a major role in distinguishing MS from controls. Lasso regression selected 44 small RNAs from three sources with just a slight reduction in AUROC (from 0.82 to 0.79) and split/join (from 10 to 12). Hence, we showcase an integrative approach with input of multi-omics data derived from multiple compartments combined with feature reduction for classification of MS.The majority (12 of 15) of top-ranked features in the reduced model were derived from MINTmap profiled plasma or cell-free CSF small RNAs, predominantly classified as tRFs, which include i-, 5′- and 3′-tRFs, cleaved at the D- and T-loops of mature tRNAs. 3′ and 5′tRFs are 14–30 nucleotides long and have previously been shown to interact with Argonaute complexes, suggesting that tRFs might have post-transcriptional regulatory potential  and may LARP7 loss-of-function variants have been associated with Alazami syndrome characterized by cognitive disability  and could therefore have similar functions as 7SK snRNA.Another top-ranked small RNA (RN7SKP118) found to be up-regulated in CSF cells of RRMS patients is a pseudogene of 7SK snRNA, which is a master transcriptional regulator of RNA polymerase II (RNAPII), involved in the production of sncRNAs such as enhancer RNAs (eRNAs), sn/snoRNAs and also mRNA . Geneticsability  and MEPCrain MRI . Hence, rain MRI . AlthougWe also identified several miRNAs in the reduced model. For example, hsa-miR-142–5p, which has previously been found in brain tissue and CSF from MS patients , serum fDespite capturing predominantly sncRNAs, we also detected protein coding and processed transcripts, in particular in plasma. Interestingly, Unc-51 Like Kinase 4 (ULK4), one of the selected protein coding transcripts in the reduced model has been proposed as a key regulator of myelination  with ULKOur integrative approach was initially also based on features from CSF cell methylome data. However, plotting of MCIA loadings from each dataset for the full model revealed that the CSF cell methylome had a minor contribution to the latent variable, L1, separating RRMS from controls (NINDC and INDC) and no CSF cell methylome features were included in the final, reduced model. Noticeably, a smoothing algorithm was applied to the PBAT methylome data, due to sparse coverage, which may have compromised the outcome, or perhaps the DNA methylation signal got diluted due to mixed cell populations in the CSF cells. It could also be that extracellular RNAs simply have greater biomarker potential for MS compared to intracellular DNA methylation. Future studies are needed for making further conclusions in this regard.In summary, we input 378 (42 × 4 × 2 + 42) profiles from cell-free CSF, CSF, plasma and PBMCs, focusing on small RNAs processed through Small-seq and MINTmap pipelines as well as CSF cell methylomes resulting in a total of 173,352 features. All datasets were derived from the same 42 individuals allowing for simultaneous integration. Although we considerably increased the input data using this approach, it is important to note that MS is a heterogeneous disease and that potentially we cover a subgroup of affected individuals. Future studies are needed to uncover whether all, some or none of the selected features separate RRMS from others in independent cohorts and a larger sample size is needed for final AUROC estimates. Here the main purpose was to trial a multi-omics integration approach for future MS biomarker development based on samples commonly collected from MS patients such as blood and CSF.


FA) in rice bran oil (RBO) and palm oil (PO) fed to growing Arbor Acre (AA) broilers. A corn-soybean meal basal diet and the basal diet supplemented with 8% RBO or PO were evaluated. A total of 72 AA male broilers  were randomly divided to 3 dietary treatments with 6 replicates of 4 broilers in each. The growth performance and the ME and FA digestibility in oils were compared with a balance experiment of broilers from d 22 to 28. The ME of the RBO diet was greater (P < 0.05) than PO diet and basal diet, and the ME of the PO diet was greater (P < 0.05) than basal diet. However, no statistical difference was observed in the ME intake of broilers fed basal, RBO, and PO diets. To maintain daily ME intake, broilers ingested more basal diet relative to other diets, resulting in increased CP intake (P < 0.01) and retention (P < 0.01) than broilers fed diets supplemented with RBO and PO. This finding resulted in greater (P < 0.01) BWG and ADG from d 22 to 28 for broilers fed the basal diet relative to other diets, but there was no difference for BWG and ADG across oil sources. However, broilers fed RBO had numerically greater BWD and ADG than those fed PO, likely because the RBO provided greater AME, AMEn, AME/GE, AMEn/GE (P < 0.01) as well as ether extract (EE) digestibility (P = 0.0536) relative to PO. The digestibility of palmitic (C16:0), stearic (C18:0), oleic (C18:1), and linoleic (C18:2) were greater (P < 0.01) in RBO than PO, which positively influenced the energy values for RBO. These results indicate RBO has greater ME and digestibility of EE and FA, which positively influenced the growth performance of AA broilers. Therefore, RBO can be used to replace PO in broiler diets.The objective of this study was to compare the digestibility of energy and fatty acids ( PO) is the most cost-effective lipid in the market and extensively used in poultry industry (SO) in adult Single Comb White Leghorn roosters. However, others reported the ME of PO ranged from 5,302 to 7,231 kcal/kg for broilers (RBO) has become more common in commercial diets in Brazil and China (MUFA) such as oleic (C18:1) (UFA) is generally beneficial to the dissolution and absorption of lipids in the digestive tract positively influencing the digestibility of fatty acids (FA) and ME   are grea (C18:1) . A high ) and ME , but an All experimental procedures related to the use of live broilers were approved by the animal care and welfare committee of the Institute of Animal Sciences, Chinese Academy of Agricultural Sciences . The code of ethical inspection was IAS 2021-221.The RBO and PO were sampled from Sunner Development Inc. . At 41°C, RBO was a brownish viscous liquid and PO (grade 3) was a yellow opaque liquid, and the PO has a higher viscosity than RBO . Other cAA) male broiler were obtained from the hatchery and housed in 3-tier cages. From d 1 to 21, broilers were provided a corn-soybean meal diet formulated to meet the requirements of Arbor Acres Broiler Nutrition Specifications , average daily gain (ADG). The AME, AMEn, and FA digestibility in test oils were determined by total collection of excreta according to the technical protocol for the determination of metabolizable energy in feed for broilers recommended by Ministry of Agriculture and Rural Affairs of P. R. China. Procedures were outlined as follows: an adaption period from 09:00 on d 22 to 16:00 on d 24 , a fast of 17 h from 16:00 on d 24 to 09:00 on d 25 to enables the complete emptying of the bird's digestive tract of feed, experimental diets were fed ad libitum starting at 09:00 on d 25 to 16:00 on d 27, and finally another fast (17 h) was started at 16:00 on d 27 to 09:00 on d 28 to collection total excreta  was measured according to the method of CP) content was determined by the method of EE) content was determined according to the method of FFA) and acid value (AV) in lipids was carried out according to standard AOCS methods . The FA composition in lipids, diets and excreta was evaluated by gas chromatography  with reference to the standard Samples were ground finely with a laboratory mill  and passed through a 0.42 mm mesh screen prior to chemical analysis. The DM content was determined according to the method of d and GEe are the GE intake  from the diet and the GE output from excreta , respectively; FI is the feed intake (kg DM); RN is the retained nitrogen (kg DM); 8.22 is the nitrogen correction factor .Dietary ME values were calculated according to the following formula:DEE) and FA were calculated according to the formula of bd is the AME  of the basal diet; AMEod is the AME  of the diet added test oil; Pbd is the proportion (%) of the basal diet in the experimental diet; Poil is the proportion (%) of test oil in the experimental diet; DCbd, DCod, and DC are the digestibility (%) of EE or FA in the basal diet, experimental diet added test oil, and test oil, respectively; the Pti is the proportional contribution (%) of EE or FA by the test oil to the experiment diet added test oil.The ME of oils and digestibility of EE . The GLM procedure of SAS 9.4  was used to analyze the differences in BWG, dietary intake, nutrient excretion and retention, nutrient digestibility, AME and AMEn among dietary treatments. Duncan's multiple comparison was used for mean separation. The AME, AMEn, AME/GE, AMEn/GE and digestibility of EE and FA in RBO and PO were compared by the TTEST procedure of SAS 9.4 . Significance was set at P = 0.0116) FI than those fed the basal diet (P < 0.05) than that of PO diet  and basal diet . Also, the ME of the PO diet were 6.15% for AME and 6.76% for AMEn greater (P < 0.05) than basal diet. The ME intake did not differ across diets, but the CP concentration of basal diet was higher than that in 2 oil diets. Other researchers have also observed that feed intake of broilers decreased with increasing dietary ME concentrations  and retention  relative to broilers fed diets with RBO and PO. These findings resulted in greater (P < 0.01) BWG and ADG from d 22 to 28 for broilers fed basal diet compared to those fed diets with RBO and PO. Accordingly, others have reported that BWG and ADG of broilers were positively correlated with the CP intake when the ME intake was identical (P < 0.05). When the AME intake was similar (P = 0.1010) for the 2 diets, but a greater digestible fat intake may supply more net energy. Therefore, the BWG and ADG of broilers increased with net energy intake when protein intake was similar (P < 0.05) than that of broilers fed RBO and PO diets. The GE content of excreta in broilers fed RBO diet was less (P < 0.05) than that of broilers fed PO diets, indicating greater undigested fat in the excreta of broilers fed PO diets. Accordingly, the EE digestibility of basal diet was greater (P < 0.01) than that of RBO and PO diets, and the EE digestibility of RBO diet was greater (P < 0.05) than that of PO diet. The RBO contains greater UFA than PO, which facilitates the absorption of FA in broilers , AMEn , AME/GE , AMEn/GE  and DEE  were greater for RBO compared to PO (C18:0) but more linoleic (C18:2) than PO, which led to the energetic values of RBO was greater than PO. High ratio of UFA to saturated fatty acids (SFA) increases the digestibility of lipid  , C18:0 , C18:1  and C18:2  were greater in RBO than PO (The digestibilities of palmitic ( than PO , which c than PO . The AME than PO . These d than PO . The dig than PO . TherefoThis experiment showed that RBO had greater AME/GE, AMEn/GE, and digestibility of EE and FAs, AME, AMEn than that of PO in AA broilers. Therefore, RBO can be used to replace PO in broiler diet. Additionally, when assessing the AME of lipid sources, the concentration of UFA should be considered. Prediction equations for the AME of RBO and PO from FA composition should be established.


Catha edulis Forsk) and are often co-abused with alcohols and polydrugs. With the increased consumption of khat and cathinones on a global scale, efforts should be channelled into understanding and minimising the excruciating effects of possible khat-drug interactions. This study aimed to determine the in vitro inhibitory effects of cathinone on CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2 and CYP3A5 and the in silico identification of their type of interactions and residues involved. The activities of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2 and CYP3A5 were examined by fluorescence based assays using recombinant cDNA-expressed human CYPs in Vivid® P450 screening kits. Cathinone reversibly inhibited CYP1A2, CYP2A6 and CYP3A5 via competitive, uncompetitive and noncompetitive modes with inhibition constant (Ki) values of 57.12, 13.75 and 23.57 µM respectively. Cathinone showed negligible inhibitory effects on CYP2B6, CYP2C8, CYP2C19, CYP2E1 and CYP2J2. Cathinone showed negligible time dependent inhibition on all 8 CYPs. Docking studies was performed on cathinone with CYP1A2, CYP2A6 and CYP3A5 following their inhibition in vitro. Cathinone is bound to a few key amino acid residues in the active sites while π-π interactions are formed in aromatic clusters of CYP1A2 and CYP3A5. These findings offer valuable reference for the use of cathinones and khat when combined with therapeutic drugs that are metabolised by CYP enzymes especially patients on medications metabolised by CYP1A2, CYP2A6 and CYP3A5.Cathinone is the psychostimulatory major active ingredient of khat (  •Active ingredient of khat plant, cathinone, inhibits human drug metabolising enzymes CYP450.•Herb-drug interactions often occur when users take herbs and medications concurrently.•Docking studies identify amino acid residues and interactions binding cathinone in CYPs.•Avoid taking khat/cathinone with drugs especially metabolised by CYP1A2, CYP2A6 and CYP3A5. Thin silico, in vitro and in vivo are very much warranted for an overall picture.The psychostimulatory effect of khat is primarily due to cathinone because the lipophilicity of cathinone is greater than cathine which favour its central nervous system (CNS) penetration, while peripheral effects are due equally to actions of cathinone and cathine N. B. . CathinoCYP isoenzymes are a family of haem-containing enzymes embedded in the lipid bilayer of endoplasmic reticulum of liver cells, responsible for drug, steroids and carcinogen metabolism In silico prediction of CYP-ligand interactions may shed light to the understanding the dynamics of CYP ligand binding recognition and affinity, prediction of inhibitors, their potency and the possible metabolites formed Despite our previous findings demonstrating that cathinone showed negligible in vitro inhibitory effects on CYP2C9, CYP2D6, and CYP3A4 22.1Cathinone or S(-)-Cathinone.HCl ((S)− 2-Amino-1-phenyl-1-propanone.hydrochloride) was procured from Lipomed AG . Acetonitrile was purchased from Fisher Scientific  while tris-base powder was obtained from Amresco® LLC . The human recombinant Vivid® kits namely CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2 and CYP3A5 was procured from Life Technologies™ . Costar black 96-well plates were obtained from Thermo Fisher Scientific .2.22 values Standard curves equations were derived by plotting fluorescence readings (RFU) produced by i) blue (3-cyano-7-hydroxycoumarin) standard with Buffer I, II and III, ii) cyan (7-hydroxy-4-trifluoromethylcoumarin) standard with Buffer II, and iii) green (fluorescein) standard against a range of concentrations  of the respective standard to obtain the standard equations and R2.3The inhibitory effects of cathinone on the catalytic activities of cDNA-expressed human CYP450 enzymes was determined using Vivid® CYP450 Screening Kits for all CYP isoforms according to manufacturer’s instructions +. Therefore, the total volume for incubation per well was 100 µL in the 96-well plate.Briefly, the assays were performed in black 96-well plates in kinetic assay mode. In each well, 40 µL of respective reaction buffers  was incubated with 50 µL of master premix , 0.3 U/ml glucose-6-phosphate dehydrogenase in 100 mM potassium phosphate at pH8.0) at room temperature for 30 min. The reaction was initiated by addition of 10 µL per well of a mixture of respective substrates and NADP+ were added, and the mixture was shaken in room temperature for 60 or 120 min based on the CYP isoform. The reaction was stopped by addition of 50 µL of 0.5 M tris-base solution and the enzyme activity was evaluated by measuring the fluorescence using Varioskan® Fluorescence Spectrophotometer  at excitation/emission wavelengths of 415/460 nm , 415/520 nm (cyan – CYP2J2) and 490/520 nm (green – CYP2C8).Following the 30 min pre-incubation, the CYP enzyme specific substrates  and 0.03 mM NADP2.450) curve. Ki values and inhibition modes were further assessed for cathinone on CYP1A2, CYP2A6 and CYP3A5 based on their respective IC50 values of less than 100 µM. Different concentrations of cathinone  was incubated with Vivid® Fluorogenic Probe Substrates EOMCC  for CYP1A2, CC  for CYP2A6 and BOMCC  for CYP3A5.Cathinone stock of 2.5 mM was prepared by dissolving cathinone in water. The assay conditions were as described above with exception that the 40 µL of buffer were substituted with 40 µL of cathinone stock at concentrations ranging from 0 to 1000 µM following two times serial dilution. The percent control activity (%) of each cathinone concentration was derived by dividing the remaining enzyme activity at each cathinone concentration, with the solvent control well (without cathinone but replaced by water) and multiplying the value with 100%. The resulting percent control activity (%) were plotted against different concentrations of cathinone to attain the half maximal inhibitory concentrations , CYP2A6 (PDB code: 2FDV) and CYP3A5 (PDB code: 6MJM) were retrieved from Protein Data Bank Open Babel GUI was employed to generate initial 3D coordinates of cathinone (O′Boyle 2011). AutoDock 4.2 2.750 values of reversible and irreversible inhibition were determined by nonlinear regression analysis using GraphPad Prism 9 for Windows . The secondary plots of cathinone concentrations against slopes of Lineweaver-Burk plots were plotted to determine the Ki values. The mode of inhibition was determined graphically from the Lineweaver-Burk plot using Microsoft Excel. All assays were carried out in triplicate and stated as mean ± SD.The IC33.12 values used in this study have been obtained from earlier studies Standard equations and R3.250 (mean ± SD) values of 17.01 (25.71 ± 34.43), 11.53 (19.22 ± 36.04) and 19.88 (21.95 ± 36.15) µM respectively as shown in nonlinear graphs , n.d., 117.5 (49.66 ± 33.48), 568.5 (40.15 ± 33.42), 162.7 (53.73 ± 33.92) µM respectively, which were more than 100 µM.Cathinone inhibited CYP1A2, CYP2A6 and CYP3A5 with ICr graphs . Cathino3.3. shows the determined IC50 without and with NADPH in pre-incubation and IC50 shift for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2 and CYP3A5. An IC50 shift of > 2 will be considered as significant TDI potency Table 13.4i values were obtained from secondary plots of each CYP isoforms. Cathinone inhibited CYP1A2, CYP2A6 and CYP3A5 via competitive mode with Ki of 57.12 µM, un-competitive mode with Ki of 13.75 µM, and noncompetitive mode with Ki of 23.57 µM as shown in the nonlinear regression – enzyme kinetics graphs  and Ki value of 17.01 (25.71 ± 34.43) and 57.12 µM; CYP2A6 via uncompetitive mode of inhibition with IC50 and Ki value of 11.53 (19.22 ± 36.04) and 13.75 µM; and CYP3A5 via noncompetitive mode of inhibition with IC50 and Ki values of 19.88 (21.95 ± 36.15) and 23.57 µM respectively  inhibited CYP2C9 via non-competitive or mixed mode with Kectively  and 3. Dectively .3 and its binding cavity fits closely with planar compounds To further understand interactions between cathinone and the major human drug metabolising CYPs, molecular docking analysis was performed. The binding energies, amino acid residues and type of interactions between cathinone and CYPs were as described in 3, catalyzes metabolism of small planar substrates besides containing three phenylalanines enabling π-π interactions with aromatic compounds and asparagine forming hydrogen bonding −1. Studies have illustrated that nicotine (phytotoxin present in many plants) is primarily metabolised by CYP2A6 and the hydrogen bonding by residue Thr305 is crucial for effective binding of pyridine scaffold of nicotine within CYP2A6 The binding pocket of CYP2A6 on the other hand is compact, with a volume of 260 ÅThe binding pocket of CYP3A4 and CYP3A5 is well- known to be relatively large and flexible, both enzymes have been recognised with more than two different ligand-binding sites From our study, cathinone, the major active ingredient present in khat, appears to only significantly inhibit CYP 1A2, CYP2A6, and CYP3A5 as compared to the other CYPs. These enzymes are among the most significantly expressed CYPs in the liver, with an expression rate of 10.3%, 3.8%, and 10.3% respectively H. F. . These Cmax  of 58.9 ± 18.8 µg/L and 71.2 ± 13.9 µg/L respectively 50 and Ki values ranging from 11.53 to 19.88 µM and 13.57 – 57.12 µM which were within 10 or 20-fold of Cmax, which indicates likelihood of drug interactions in spite of the milder risks.Earlier studies by Widler et al. showed that the maximal plasma concentration of cathinone was 127 ± 53 ng/ml) after 127 ± 30 min, with area under the plasma concentration – time curve (AUC) from 0 to 9 h of 415 ± 207 ng/ml per hour and terminal elimination half-life of 260 ± 102 min Khat use have been found to hinder response to antipsychotic In vivo studies could assist the results by measuring the concentrations of main metabolite of a provided test drug in control and exposure groups. A decrease in the drug metabolites either from the test drug and khat exposure group could be used to distinguish whether khat causes an inhibition or otherwise, affecting the CYPs metabolism directly.Further in vivo studies using animal models are warranted to investigate the effects of cathinone, cathine and other phytochemicals in khat plant on CYP gene expressions which is ongoing in our laboratory besides to validate the clinical relevance via human subjects to grasp effects of khat on all CYP isoforms including CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2 and CYP3A5. Besides CYPs, there are other detoxification systems such as aldehyde oxidase (AOX), which is a non-CYP metabolic enzymes and cytosolic drug metabolising enzyme expressed in the human liver which is similar to CYPs which contributes to oxidation but act in the absence of NADPH cofactor In silico studies on interaction of other khat constituents for instance cathine, tannins, cathedulins, just to name a few, with CYPs are warranted to visualise the possible interactions that is worthy to be explored.Despite the evidence that cathinone inhibited a few important drug metabolising CYPs in vitro, the in vivo processes can be entirely divergent. The limitations of this study includes; (1) fluorescent substrates may interact with CYP differently than classic drug substrates which is why they are used as initial screening tools only; (2) recombinant CYP is very simple compared to microsomes or hepatocytes; and (3) CYP inhibition must be confirmed using more relevant drug substrates with in vitro ; (4) in vivo clinical studies. Moreover, in vitro study could not encompass pharmacokinetic factors such as gastric digestion, absorption of cathinone and first-pass effect in human especially if cathinone is administered orally, the cathinone concentration will be greatly reduced before reaching the systemic circulation. Worth mentioning is that the present study uses only one substrate for each CYP isoform which could be improved by using different substrates to compare the possible interactions. Cathinone is often blamed for the dependence and/or addiction issue ascribed to khat chewing 5This study is an initial screening that must be confirmed using more accurate models. Cathinone inhibited CYP1A2, CYP2A6 and CYP3A5 only but showed negligible in vitro inhibitory effects on CYP2B6, CYP2C8, CYP2C19, CYP2E1 and CYP2J2. CYP1A2, CYP2A6 and CYP3A5 are among the major human drug metabolising enzymes, responsible for metabolising various commonly used medications including antibiotics, immunosuppressive drugs, anticoagulants, anticancer drugs as well as painkillers. Cathinone inhibited CYP1A2, CYP2A6 and CYP3A5 via non-competitve and uncompetitive mixed mode. Docking outcome demonstrated the competitive inhibition of cathinone on CYPs were similar to prior research utilizing CYPs’ inhibitors, that binds closely to the haem group and surrounding residues. Conserved amino acid residues were also involved in cathinone-CYPs binding. In addition, hydrophobic, hydrogen bond and π-stacking interactions are among the type of interactions contributing to inhibitory activity of cathinone on CYP1A2, CYP2A6 and CYP3A5. Our study demonstrates that khat consumption may result in inhibition of CYP1A2, CYP2A6, and CYP3A5 through its major active component, cathinone, with potential clinically-significant implications for users who may be taking prescribed medications that are metabolised through these pathways. It would therefore be prudent for healthcare professionals to be aware of these potential interactions during their prescribing and treatment of khat users, either by advising abstinence or adjusting their prescribing accordingly.Sharoen Yu Ming Lim: Conceptualization, Formal analysis, Investigation, Data curation, Writing – original draft, Writing – review & editing, Visualization, Project administration. Jason Siau Ee Loo: Software, Formal analysis, Writing – review & editing, Visualization, Supervision. Mustafa Alshagga: Conceptualization, Methodology, Resources, Supervision, Funding acquisition Mohammed Abdullah Alshawsh: Conceptualization, Resources. Chin Eng Ong: Conceptualization, Methodology Yan Pan: Conceptualization, Methodology, Validation, Resources, Writing – review & editing, Supervision.University of Nottingham Malaysia [grant number 54-ER-0-744019].This work was supported by the None.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The derangement of CSF circulation impacts the functions of the glymphatic-lymphatic system (G-Ls), which regulates solute trafficking and immune surveillance in the CNS. The G-Ls failure leads to the dysregulation of clearance of waste molecules in the brain and to an altered CNS immune response. The imaging features of dilated perivascular spaces imply the impairment of the G-Ls. We report on the case of a patient with primary progressive multiple sclerosis and dilatation of perivascular spaces, who transiently improved after CSF shunt diversions. The underlying mechanisms remain to be determined and at this stage, it is not possible to link CSF diversion to an effect on MS pathology. However, this observation provides the rationale to incentivize research in the largely unknown area of CSF dynamic disturbances on G-Ls failure and ultimately in neurodegeneration. The glymphatic-lymphatic system (G-Ls) regulates solute trafficking and immune surveillance in the CNS, sub-serving the flow of CSF from the subarachnoid spaces into the perivascular spaces and subsequently into the interstitium, with the aquaporin-4 water channels running it  , performed in 2010, revealed diffuse hyperintense T2 nodular lesions affecting the paraventricular and subcortical white matter; at least two of these lesions showed gadolinium enhancement in T1-weighted images. A coeval MRI of the spinal cord showed nodular lesions involving the dorsal columns, at the C2 and C3 levels. The blood tests were within physiological parameters, while the neurophysiological examination revealed changes in visual evoked potential and somatosensory evoked potential from the left median nerve. Oligoclonal IgG bands at isoelectric focusing occurred in the CSF. The clinical and paraclinical findings led to the diagnosis of MS. Methylprednisolone provided the patient with some improvement. Immunomodulatory treatments with interferon-beta and glatiramer acetate were started and later discontinued due to intolerance. After 16 months of continuous neurological impairment, the patient was diagnosed with primary progressive MS. An MRI performed during the same period revealed a stable picture. She was voluntarily tested in another hospital for cerebrospinal venous insufficiency in 2012. A severe cerebrospinal venous insufficiency was diagnosed, and she underwent percutaneous transluminal angioplasty at the level of the internal jugular veins. She had about 2 years of clinical benefit after the procedure, which paralleled normal anatomic and functional conditions of jugular veins according to the echo color Doppler examination and stability of MS lesions at MRI. Subsequently, her neurological condition deteriorated gradually. Sonographic findings suggested a recurrence of chronic cerebrospinal venous insufficiency (2014). At the age of 38, the patient came under our observation  with a dAware of the anecdotal nature, potential placebo effect, and possibly unrelated and unknown factors, this case suggests the effect of CSF diversions on the MS patient's clinical improvements. The mechanisms and factors underpinning the CSF shunt-mediated improvements of the MS symptoms remain to be determined, at this stage. CSF diversion, decongesting perivascular spaces, may have ameliorated glymphatic functioning and interstitial removal of proinflammatory molecules from CSF and in so doing, downregulating local inflammation. This is in line with the Louveau et al. hypothesis that the modulation of CSF drainage through the meningeal lymphatic vasculature might reduce the quantity of CNS antigens entering the related lymph nodes, resulting in the downregulation of the autoimmune response  for the publication of any potentially identifiable images or data included in this article.AS, BP, and PG have made substantial contribution to the conception of the study. PG, FL, BP, AR, GL, CN, and GD have drafted the work or substantially revised it. All authors contributed to the article 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.Videos of a patient with primary progressive multiple sclerosis imaging features of dilated perivascular spaces who underwent cerebrospinal fluid diversions.


The way information about potential harms of trial intervention is shared within participant information leaflets (PILs) varies widely and can cause subjective ‘nocebo’ harms. This study aimed to develop principles to improve the composition of information about potential trial intervention benefits and harms within PILs so that variability and avoidable harms are reduced.We conducted a two-round modified online Delphi survey, followed by a consensus meeting. For the first round of the survey, 27 statements were developed based on previous research and relevant guidance from the UK, the USA and the World Health Organization. Participants included members from each of the following stakeholder groups: patient and public representatives, research ethics committee members, industry representatives, medico-legal experts, psychologists and trial managers. Each participant was asked to rate their degree of agreement or disagreement with each statement on a 9-point Likert scale. In the second round, participants were invited to reappraise their ratings after reviewing the results of the first round. Finally, two members from each stakeholder group participated in a meeting to confirm those statements for which there was agreement.Two hundred and fifty participants completed round 1, and 201 participants completed round 2. In round 1, consensus was reached for 16 statements. In round 2, consensus was reached for an additional three statements. The consensus meeting confirmed the survey results and consolidated the statements. This process resulted in seven principles: (1) all potential harms of a given intervention should be listed, (2) all potential harms should be separated into serious and less serious, (3) it must be made explicit that not all potential harms are known, (4) all potential benefits should be listed, (5) all potential benefits and harms need to be compared with what would happen if the participant did not take part in the trial, (6) suitable visual representations should be added where appropriate and (7) information regarding potential benefits and harms should not be presented apart by one or more pages.Our modified Delphi process successfully generated seven principles that can and should be used to guide how information is conveyed to patients in information leaflets regarding potential trial benefits and harms.The online version contains supplementary material available at 10.1186/s13063-022-06780-1. The potential benefits and risks of trial interventions are not communicated to patients in a consistent way. In a recent analysis of 33 participant information leaflets (PILs) used in trials conducted by the National Institute of Health Research (NIHR) in the UK , the wayThe way information about potential trial treatment harms is communicated also poses an under-recognised ethical issue. The requirement of autonomy demands that trial participants be informed about all potential harms. However, if the way information about potential harms is conveyed causes harm, the ethical responsibility of non-maleficence may be violated. As far as we are aware, the ethical debate related to how information about potential trial treatment harms should be shared focuses on autonomy and neglects non-maleficence .At present, no guidance is available that explains how to present information about potential trial benefits and harms in a way that respects the need to share information (respecting autonomy) and that is balanced (less likely to induce nocebo effects). Therefore, every principal investigator must negotiate their own method for sharing information about trial benefits and harms in a balanced way. This leads to the heterogeneity and increased risk of nocebo effects noted above. Ultimately, these biases in primary studies may lead to biases in meta-analyses and distort evidence of intervention effects that may affect judgements about effectiveness, cost-effectiveness and efficiency.This study aimed to develop consensus-based principles to guide how information about trial intervention harms and benefits should be shared with patients so that unnecessary variation and harm is minimized.Following a published protocol , we usedPrinciples and examples from our review of UK PILs Extracted principles and examples from a random sample of Drug Facts Boxes Statements from official guidance about presenting trial benefits and harms in PILs from within the UK  ) and intWe generated a list of potential information about benefits and harms from three sources that background research suggested to be important:The long list derived from these sources was deduplicated and piloted for face validity by our co-applicant group and patient and public representatives.There is currently no standard method for determining sample size calculations for Delphi studies . The criTo maximise responses across stakeholder groups and to achieve diverse representation, we monitored the survey responses and sent additional reminders to groups that had fewer responses. The total number of respondents per stakeholder group was reviewed between rounds.Public and patient representatives: Our patient and public representative (Jennifer Bostock) and advisory board member (Jono Broad) helped us identify these representatives from their networks, including Pain UK and People in Research, NIHR, the James Lind Alliance, the NIHR Centre for Engagement and Dissemination and health literacy groups [y groups .Research ethics committee members and other approval staff: The principal investigator and co-applicants have contacts at the HRA who put the study team in touch with interested ethics committee members and chairs.Industry : Experts were identified with help from our industry partners  and with input from the advisory group .Applied researchers, psychologists and risk communicators: These were identified by the principal investigator and co-applicants, who have extensive networks of psychologists with relevant expertise. The study team targeted psychologists with a range of relevant expertise, especially in risk communication and behavioural science.Research nurses, clinical trial managers and trialists: Researchers were identified via the UK Clinical Research Collaboration (UKCRC), the Registered Centre for Trials Research at Cardiff University and the UK Trial Managers’ Network (UKTMN).A group of stakeholders was identified from the contact lists, networks of co-applicants and patient and public representatives. The survey was only available in English due to the time limitations of the study. Stakeholders included representatives from each of the five following sources:Because of the different medico-legal frameworks in different countries, we restricted our stakeholders to those based in the UK.Survey data were collected using Qualtrics . ParticiFollowing the methods used in a related study , our DelAnalyses were conducted on fully anonymised Qualtrics survey data. Descriptive statistics were used to summarise the results from each round. For each item, the distribution of scores was summarised by stakeholder groups alongside the total number of participants who scored the item. Participants were instructed to rate each item independently even if items appeared similar.The participant flow diagram Fig.  outlinesIn round 1, participants were presented with 27 statements across four sections. The first section included four hypothetical scenarios that described (i) a study participant being given excessive information about possible harms, (ii) not enough information about possible harms, (iii) a comparison of intervention with what would occur if a participant took nothing, and (iv) one scenario about positive framing. The second and third sections both related to describing the potential harms and benefits of a clinical trial. The fourth section included statements concerning the order and layout of benefits and harms in the participant leaflet. Participants were asked to rate their agreement or disagreement with the statements using a scale from one to nine, where one corresponds to ‘strongly agree’ and nine corresponds to ‘strongly disagree’. Participants were also given the opportunity to share free-text comments where they could provide reasons for their answers and propose further items for inclusion in round 2. Round 1 terminated when at least 20 participants across each of the stakeholder groups responded.The research team then tabulated an overview of the total number of participants and each stakeholder group. For purposes of analysis, the scale was divided into clusters representing agreement (scoring 1–3), indecision (scoring 4–6) and disagreement (scoring 7–9)  for those items which exhibited no consensus and to confirm those items for which there was agreement  was achieved to progress to round 2. Two hundred one participants took part in round 2. The overall attrition rate between round 1 and round 2 was 19.6%, with the highest rate of 37.5% for industry stakeholders and the lowest rate of 8.3% for research ethics committee members and other approvals staff. Partial responses were included in the analysis for both rounds.Two hundred fifty stakeholders responded to the invitation email, gave informed consent and completed round 1  from the Delphi survey  but not overall. After discussion, the consensus meeting attendees agreed that this statement was redundant, and that potential benefits should always be listed.The statement, Information about potential benefits and harms should be mentioned in more than one place in the leaflet, reached consensus amongst applied researchers and clinical trial professionals but not overall. The consensus meeting attendees agreed that PILs should be clear and concise and that anything that makes them longer and more complicated than necessary should be avoided. Repetition of benefits and harms in more than one place would not add any value, so the group agreed to leave this statement out.The statement, A complete (detailed) description of the potential harms (and the likelihood of each harm) should be provided in a table in an appendix, reached consensus amongst the research ethics committee members and ‘near’ consensus amongst the public, patients and their advocates and clinical trial professions groups. The consensus group meeting attendees recognised that some potential trial participants might want condensed information on potential harms while others will prefer more detailed information that an appendix could provide. In the end, the meeting attendees agreed to leave the statement out.The statement, Drug fact boxes... divide harms into serious and non-serious. This way of presenting harms is helpful, reached consensus amongst the public, patients and their advocates and applied researchers. The consensus group, while acknowledging that drug fact boxes are a useful clinical tool, found that there was a level of uncertainty regarding the generalizability of the tool to non-drug treatments and trials. Consequently, a consensus was not reached.The statement, The consensus meeting also assisted in resolving some differences between responses across the stakeholder groups in previous rounds and in harmonising some of the statements in several areas:Once full consensus was reached on the statements, the participants spent the remainder of the in-person meeting discussing aesthetic modifications to the final set of principles, including the possibility of grouping similar principles together to remove any repetition. The final principles, together with the survey statements that they are based on, are shown in Table We were able to identify seven principles to guide how information is shared regarding potential trial intervention benefits and harms. The principles are based on consensus exercises involving a wide range of stakeholders.Guidance for sharing information about potential trial benefits and harms within PILs is under-researched. An exception may be ‘Drug Facts Boxes’, which were developed to improve patient understanding of drug benefits and harms . HoweverDespite the large sample for this type of study (more than double what we planned for), the stakeholders may not have been representative of all relevant parties. We believe that the high retention rates and the clear consensus on most items mitigate the impact of this potential problem.The principles developed by our exercise can now be used to design the relevant sections of PILs. PILs developed according to these principles should be rigorously compared with other PILs to check whether they reduce nocebo effects or improve recruitment and retention rates. This could be straightforwardly achieved by using ‘studies with a trial’ (‘SWATs’).in principle between an effect that is harmful and an effect that is beneficial [The seven core principles described in this study can also be used to inform future HRA guidance on sharing information about potential trial treatment benefits and harms. Importantly, the principles revealed by the rigorous process of this study are broadly in line with current HRA guidance; however, there are some important differences. For example, whereas current relevant HRA guidance is brief (which could be a cause of the variability) , our guineficial . In factneficial . This isneficial . For exaneficial . TherefoIn addition, practical guidance on how to implement the principles could be produced. This would serve to reduce variability in the way trial benefits and harms are described. Clear guidance could reduce the time spent by those who design PILs and ethics committees who review them. To be implemented, additional research would need to be done about the differences between drug and non-drug trials. Potential harms of drugs are usually listed comprehensively , but for non-drug trials, additional research is often required to derive a complete list of potential harms.Our study also contributes to recent ethical research, which has found that research ethics committees are overly focused on one interpretation of the principle of autonomy, leading to an over-emphasis on describing potential harms . The priThese principles should be adapted for at least two related areas: clinical practice and instruction about how to verbally communicate information about potential benefits and harms. In clinical practice, healthcare practitioners could use a version of these principles to describe potential benefits and harms to their patients. Relatedly, a training package that explains how these principles should be explained verbally could be developed for research nurses and others who take informed consent. Our principles could also be tested in different countries that do not share the same medico-legal context as the UK.Clear consensus about seven principles to guide the way potential benefits and harms or trial treatments are described within patient information leaflets was reached from a wide range of stakeholders. These principles can now be implemented to design and evaluate patient information leaflets to reduce variability in the way in which information about potential benefits and harms of trial treatments are described.Additional file 1. CREDES Checklist.Additional file 2. Delphi survey instruction.Additional file 3. Full round 1 results.Additional file 4. Summary round 1 results.Additional file 5. Full round 2 results.Additional file 6. Consensus meeting statements.Additional file 7. Round 1 & 2 free-text comments.Additional file 8. Round 1 free-text comments.Additional file 9. Round 2 free-text comments.Additional file 10. Consensus meeting description.Additional file 11. Final set of principle statements.