Comparison of CRISPR-MAD7 and CRISPR-Cas9 for Gene Disruptions in Komagataella phaffii.

CRISPR (clustered regularly interspaced short palindromic repeats)-based technologies are powerful, programmable tools for site-directed genome modifications. After successful adaptation and efficient use of CRISPR-Cas9 for genome engineering in methylotrophic yeast Komagataella phaffii, a broader variety of employable endonucleases was desired to increase the experimental flexibility and to provide alternatives in case there are specific legal restrictions in industrial research due to the intellectual property rights (IPRs) of third parties. MAD7, an engineered Class 2 Type V Cas nuclease, was promoted as a royalty-free alternative for academic and industrial research and developed by Inscripta (Pleasanton, CA, USA). In this study, for the first time, CRISPR-MAD7 was used for genome editing in K. phaffii with a high gene-editing rate (up to 90%), as demonstrated for the three targeted genes coding for glycerol kinase 1 (GUT1), red fluorescence protein (DsRed), and zeocin resistance gene (Sh ble). Additionally, the genome-editing efficiencies of the CRISPR-MAD7 and CRISPR-Cas9 systems were systematically compared by targeting 259 kinase genes in K. phaffii. In this broad testing, the CRISPR-Cas9 had a higher genome-editing rate of about 65%, in comparison to the applied CRISPR-MAD7 toolbox (about 23%).

The WOX Genes from the Intermediate Clade: Influence on the Somatic Embryogenesis in Medicago truncatula.

Transcription factors from the WOX family are well-known regulators of cell proliferation and differentiation in plants. Herein, we focused on several WOX genes from the intermediate clade and checked their impact on somatic embryogenesis using the model legume object Medicago truncatula. As a result, we show that MtWOX9-1 overexpression not only stimulates somatic embryogenesis in the embryogenic M. truncatula line, as it was shown previously, but can also induce somatic embryogenesis in the non-embryogenic line. Other intermediate clade WOX, including the close paralog of MtWOX9-1, as well as WOX11 homologs, did not have any significant impact on somatic embryogenesis in our in vitro cultivation system. Together, our results give new information about the diversity of the WOX family proteins and their specific functions. These data can be used for the search of new regeneration stimulators.

Alpha2-Adrenergic Receptors as a Pharmacological Target for Spike-Wave Epilepsy.

Spike-wave discharges are the hallmark of idiopathic generalized epilepsy. They are caused by a disorder in the thalamocortical network. Commercially available anti-epileptic drugs have pronounced side effects (i.e., sedation and gastroenterological concerns), which might result from a low selectivity to molecular targets. We suggest a specific subtype of adrenergic receptors (ARs) as a promising anti-epileptic molecular target. In rats with a predisposition to absence epilepsy, alpha2 ARs agonists provoke sedation and enhance spike-wave activity during transitions from awake/sedation. A number of studies together with our own observations bring evidence that the sedative and proepileptic effects require different alpha2 ARs subtypes activation. Here we introduce a new concept on target pharmacotherapy of absence epilepsy via alpha2B ARs which are presented almost exclusively in the thalamus. We discuss HCN and calcium channels as the most relevant cellular targets of alpha2 ARs involved in spike-wave activity generation.

MtWOX2 and MtWOX9-1 Effects on the Embryogenic Callus Transcriptome in Medicago truncatula.

WOX family transcription factors are well-known regulators of plant development, controlling cell proliferation and differentiation in diverse organs and tissues. Several WOX genes have been shown to participate in regeneration processes which take place in plant cell cultures in vitro, but the effects of most of them on tissue culture development have not been discovered yet. In this study, we evaluated the effects of MtWOX2 gene overexpression on the embryogenic callus development and transcriptomic state in Medicago truncatula. According to our results, overexpression of MtWOX2 leads to an increase in callus weight. Furthermore, transcriptomic changes in MtWOX2 overexpressing calli are, to a large extent, opposite to the changes caused by overexpression of MtWOX9-1, a somatic embryogenesis stimulator. These results add new information about the mechanisms of interaction between different WOX genes and can be useful for the search of new regeneration regulators.

Social Behavioral Deficits in Krushinsky-Molodkina Rats, an Animal Model of Audiogenic Epilepsy.

In clinical practice, epilepsy is often comorbid with the autism spectrum disorders (ASDs). This warrants a search of animal models to uncover putative overlapping neuronal mechanisms. The Krushinsky-Molodkina (KM) rat strain is one of the oldest inbred animal models for human convulsive epilepsies. We analyzed the behavioral response of adult seizure-naive KM males in three-chambered tests for social preference. We found that a presence of social stimuli (encaged unfamiliar Wistar rats of the same age and sex) evoked a reduced or reversed exploratory response in freely moving KM individuals. The epilepsy-prone rats demonstrated remarkably shortened bouts of social contacts and displayed less locomotion around the stranger rat-containing boxes, together with a pronounced freezing response. The decrease in social preference was not due to a general decrease in activity, since relative measures of activity, the index of sociability, were decreased, too. The susceptibility to audiogenic seizures was verified in the KM cohort but not seen in the control Wistar group. We propose the KM rat strain as a new animal model for comorbid ASD and epilepsy.

Influence of early social isolation on general activity and spatial learning in adult WAG/Rij rats.

The study identifies the critical period of early ontogeny, during which social factors have the greatest influence on the operant behavior with positive reinforcement in adult WAG/Rij rats. Individual social isolation of rats from dam and siblings was performed daily for 3 h during postnatal day (PND) 2-8, 9-15, and 16-22. General activity and water consumption were examined using the IntelliCage (IC) in adulthood. The operant behavior training was performed in four consecutive sessions: free exploration of the IC environment (adaptation), learning to retrieve water by nosepoking (nosepoke adaptation), spatial learning to retrieve water in the specific corner (place learning), and retraining with a change of a place preference (reversal learning). Social isolation during PND16-22 led to the greatest behavioral changes in all sessions of the experiment. These rats were more active, consumed more water, demonstrated a higher ratio of visits with drinking to the total number, and relearned faster after changing the location of the rewarded corner. Thus, the postnatal period between days 16 and 22 in WAG/Rij rat pups is more sensitive to social isolation for change of adaptive behavior in the IC in adulthood.

Electrophysiological and Behavioral Evidence for Hyper- and Hyposensitivity in Rare Genetic Syndromes Associated with Autism.

Our study reviewed abnormalities in spontaneous, as well as event-related, brain activity in syndromes with a known genetic underpinning that are associated with autistic symptomatology. Based on behavioral and neurophysiological evidence, we tentatively subdivided the syndromes on primarily hyper-sensitive (Fragile X, Angelman) and hypo-sensitive (Phelan-McDermid, Rett, Tuberous Sclerosis, Neurofibromatosis 1), pointing to the way of segregation of heterogeneous idiopathic ASD, that includes both hyper-sensitive and hypo-sensitive individuals. This segmentation links abnormalities in different genes, such as FMR1, UBE3A, GABRB3, GABRA5, GABRG3, SHANK3, MECP2, TSC1, TSC2, and NF1, that are causative to the above-mentioned syndromes and associated with synaptic transmission and cell growth, as well as with translational and transcriptional regulation and with sensory sensitivity. Excitation/inhibition imbalance related to GABAergic signaling, and the interplay of tonic and phasic inhibition in different brain regions might underlie this relationship. However, more research is needed. As most genetic syndromes are very rare, future investigations in this field will benefit from multi-site collaboration with a common protocol for electrophysiological and event-related potential (EEG/ERP) research that should include an investigation into all modalities and stages of sensory processing, as well as potential biomarkers of GABAergic signaling (such as 40-Hz ASSR).

Spike-Wave Seizures, NREM Sleep and Micro-Arousals in WAG/Rij Rats with Genetic Predisposition to Absence Epilepsy: Developmental Aspects.

The current study was done in Wistar Albino Glaxo Rijswijk (WAG/Rij) rats, which are genetically prone to develop spontaneous spike-wave discharges (SWDs) and are widely used as a genetic model of absence epilepsy. Here, we examined functional links between sleep and spike-wave epilepsy in aging WAG/Rij rats using advanced techniques of EEG analysis. SWDs, periods of NREM sleep and micro-arousals were automatically detected in three-channel epidural EEG recorded in freely moving WAG/Rij rats consequently at the age 5, 7 and 9 months. We characterized the developmental profile of spike-wave epilepsy in drug-naïve WAG/Rij rats and defined three epi-phenotypes-severe, mild and minor epilepsy. Age-related changes of SWDs were associated with changes in NREM sleep. Several signs of NREM sleep fragmentation were defined in epileptic WAG/Rij rats. It seems that spike-wave epilepsy per se promotes micro-arousals during NREM sleep. However, subjects with a higher number of micro-arousals (and NREM sleep episodes) at the age of 5 months were characterized by a reduction of SWDs between 5 and 7 months of age.

Whisker trimming during infanthood modifies the development of spike-wave discharges and behavioral sequences in IntelliCage impulsivity paradigm in adult WAG/Rij rats.

Whisker system in rats undergoes rapid development during the first postnatal weeks. Neonatal whisker trimming increases excitability in the somatosensory cortex and affects exploratory behavior at adult ages. WAG/Rij rats are genetically predisposed to develop absence seizures in adulthood, and whisker trimming during three postnatal weeks aggravates epileptic activity in these rats. It is assumed that behavioral performance in adult WAG/Rij rats is influenced (1) by absence epilepsy and (2) by whisker trimming during the short period around the onset of active whisker movements, PN9-16. We examined the effect of whisker trimming in WAG/Rij rats during PN9-16 on spike-wave discharges (SWD, EEG hallmark of absence epilepsy). We found that 77% of WAG/Rij rats showed pronounced SWD (epileptic phenotype), and the rest did not (non-epileptic phenotype). At the age of 5 m, epileptic trimmed rats showed more SWD than epileptic control rats. Age-related increase of SWD was found only in the control group, suggesting that whisker trimming during PN9-16 led to an earlier maturation of SWD. Goal-directed behavior was examined in all rats at the age of 4-4.5 m using IntelliCage impulsivity paradigm. In order to optimize the analysis of behavioral data, we combined several Python packages into a single processing pipeline. Early life whisker trimming altered behavioral sequences and strategy of exploration in adulthood, suggesting reduced whisker sensitivity in the trimmed rats. Epileptic WAG/Rij rats at 4-4.5 months showed only a slight learning impairment during later stages of IntelliCage impulsivity paradigm, which may be associated with the early stage of development of SWD.

Linking the FTO obesity rs1421085 variant circuitry to cellular, metabolic, and organismal phenotypes in vivo.

Variants in FTO have the strongest association with obesity; however, it is still unclear how those noncoding variants mechanistically affect whole-body physiology. We engineered a deletion of the rs1421085 conserved cis-regulatory module (CRM) in mice and confirmed in vivo that the CRM modulates Irx3 and Irx5 gene expression and mitochondrial function in adipocytes. The CRM affects molecular and cellular phenotypes in an adipose depot-dependent manner and affects organismal phenotypes that are relevant for obesity, including decreased high-fat diet-induced weight gain, decreased whole-body fat mass, and decreased skin fat thickness. Last, we connected the CRM to a genetically determined effect on steroid patterns in males that was dependent on nutritional challenge and conserved across mice and humans. Together, our data establish cross-species conservation of the rs1421085 regulatory circuitry at the molecular, cellular, metabolic, and organismal level, revealing previously unknown contextual dependence of the variant's action.

Reviewing Evidence for the Relationship of EEG Abnormalities and RTT Phenotype Paralleled by Insights from Animal Studies.

Rett syndrome (RTT) is a rare neurodevelopmental disorder that is usually caused by mutations of the MECP2 gene. Patients with RTT suffer from severe deficits in motor, perceptual and cognitive domains. Electroencephalogram (EEG) has provided useful information to clinicians and scientists, from the very first descriptions of RTT, and yet no reliable neurophysiological biomarkers related to the pathophysiology of the disorder or symptom severity have been identified to date. To identify consistently observed and potentially informative EEG characteristics of RTT pathophysiology, and ascertain areas most worthy of further systematic investigation, here we review the literature for EEG abnormalities reported in patients with RTT and in its disease models. While pointing to some promising potential EEG biomarkers of RTT, our review identify areas of need to realize the potential of EEG including (1) quantitative investigation of promising clinical-EEG observations in RTT, e.g., shift of mu rhythm frequency and EEG during sleep; (2) closer alignment of approaches between patients with RTT and its animal models to strengthen the translational significance of the work (e.g., EEG measurements and behavioral states); (3) establishment of large-scale consortium research, to provide adequate Ns to investigate age and genotype effects.

Automatic wavelet-based assessment of behavioral sleep using multichannel electrocorticography in rats.

PURPOSE: During the last decade, the reported prevalence of sleep-disordered breathing in adults has been rapidly increasing. Therefore, automatic methods of sleep assessment are of particular interest. In a framework of translational neuroscience, this study introduces a reliable automatic detection system of behavioral sleep in laboratory rats based on the signal recorded at the cortical surface without requiring electromyography. METHODS: Experimental data were obtained in 16 adult male WAG/Rij rats at the age of 9 months. Electrocorticographic signals (ECoG) were recorded in freely moving rats during the entire day (22.5 ± 2.2 h). Automatic wavelet-based assessment of behavioral sleep (BS) was proposed. The performance of this wavelet-based method was validated in a group of rats with genetic predisposition to absence epilepsy (n=16) based on visual analysis of their behavior in simultaneously recorded video. RESULTS: The accuracy of automatic sleep detection was 98% over a 24-h period. An automatic BS assessment method can be adjusted for detecting short arousals during sleep (microarousals) with various duration. CONCLUSIONS: These findings suggest that automatic wavelet-based assessment of behavioral sleep can be used for assessment of sleep quality. Current analysis indicates a temporal relationship between microarousals, sleep, and epileptic discharges in genetically prone subjects.

Active avoidance learning in WAG/Rij rats with genetic predisposition to absence epilepsy.

Neurological disorders are often accompanied by impairment of memory, attention deficit that cause learning difficulties. Better understanding of learning problems in neurological patients might help to improve the quality of their life. Here we studied the character of fear-based associative learning using the standard active avoidance test in WAG/Rij rats with genetic predisposition to absence epilepsy. Electroencephalographic properties of spike-wave seizures (i.e., hallmarks of absence epilepsy) were examined at the age of 5 and 7 m. Around 24 % of rats did not express epileptic activity despite genetic predisposition. In the active avoidance test, 6 m old rats with the epileptic phenotype needed more trails to obtain the first avoidance than non-epileptic rats, but showed the same number of avoidances to reach the learning criterion. The total time of spike-wave activity positively correlated with the outcomes of avoidance performance only in subjects with severe epilepsy, but not in subjects with mild epilepsy. In order to evaluate early sensory (epigenetic) factors governing cognitive comorbidities in adult WAG/Rij rats, we performed bilateral whisker trimming during PN1-8 and PN9-16. This led to a quicker development of SWD, but did not influence cognitive abilities at the age of 6 m. In summary, epileptic WAG/Rij rats had difficulties with executive functions before the first avoidance of adverse stimulus, rather than impairment of memory after the first avoidance. Our data assume that cognitive comorbidities in epileptic WAG/Rij rats primarily may relate to executive deficit during the initial stage of avoidance test and secondary - to impairment of short-term memory. This fits well to outcomes of clinical studies in patients with generalized epilepsy.

Sensory evoked potentials in patients with Rett syndrome through the lens of animal studies: Systematic review.

OBJECTIVE: Systematically review the abnormalities in event related potential (ERP) recorded in Rett Syndrome (RTT) patients and animals in search of translational biomarkers of deficits related to the particular neurophysiological processes of known genetic origin (MECP2 mutations). METHODS: Pubmed, ISI Web of Knowledge and BIORXIV were searched for the relevant articles according to PRISMA standards. RESULTS: ERP components are generally delayed across all sensory modalities both in RTT patients and its animal model, while findings on ERPs amplitude strongly depend on stimulus properties and presentation rate. Studies on RTT animal models uncovered the abnormalities in the excitatory and inhibitory transmission as critical mechanisms underlying the ERPs changes, but showed that even similar ERP alterations in auditory and visual domains have a diverse neural basis. A range of novel approaches has been developed in animal studies bringing along the meaningful neurophysiological interpretation of ERP measures in RTT patients. CONCLUSIONS: While there is a clear evidence for sensory ERPs abnormalities in RTT, to further advance the field there is a need in a large-scale ERP studies with the functionally-relevant experimental paradigms. SIGNIFICANCE: The review provides insights into domain-specific neural basis of the ERP abnormalities and promotes clinical application of the ERP measures as the non-invasive functional biomarkers of RTT pathophysiology.

Developmental milestones and behavior of infant rats: The role of sensory input from whiskers.

Developmental milestones are behavioral and physical skills which are considered as markers of neurodevelopment. In rodents, sensory input from whiskers plays a crucial role in development of brain functions. Development of whisker system in rats includes the early period of passive whisker touch (PN1-8) before the onset of coordinated whisker movements which underlie active sensing. Inasmuch as transitioning from passive to active sensing requires a strong sensorimotor integration, we assume that the effect of whisker deprivation during the period of passive touch is unfavorable for neurodevelopment, but deprivation after the onset of active sensing might elicit less harmful effect due to compensatory neuroplalstic changes. Here we examined the effect of complete whisker trimming (WT) in WAG/Rij rats during PN1-8 and PN9-16 (active sensing) on achieving developmental milestones (e.g., eyelid opening, walking, self-grooming, rearing activity, physical maturation of forelimbs), locomotor activity and body weight. Control groups underwent sham trimming during the same periods. WT during PN1-8 caused a delay in achieving all investigated milestones, but WT during PN9-16 delayed only self-grooming. Both WT/sham trimming during PN9-16 caused a delay in explorative behavior, but accelerated self-grooming. These changes are likely to link with the effect of manipulations during PN9-16 in previously unhandled pups, but not specifically with WT. In general, developmental milestones appeared to be an informative tool to access neurodevelopment in rat pups and might have a translational value for studying developmental disorders during early life.

Mass Difference Maps and Their Application for the Recalibration of Mass Spectrometric Data in Nontargeted Metabolomics.

Modern high-resolution mass spectrometry provides the great potential to analyze exact masses of thousands of molecules in one run. In addition, the high instrumental mass accuracy allows for high-precision formula assignments narrowing down tremendously the chemical space of unknown compounds. The adequate values for a mass accuracy are normally achieved by a proper calibration procedure that usually implies using known internal or external standards. This approach might not always be sufficient in cases when systematic error is highly prevalent. Therefore, additional recalibration steps are required. In this work, the concept of mass difference maps (MDiMs) is introduced with a focus on the visualization and investigation of all the pairwise differences between considered masses. Given an adequate reference list of sufficient size, MDiMs can facilitate the detection of a systematic error component. Such a property can be potentially applied for spectral recalibration. Consequently, a novel approach to describe the process of the correction of experimentally derived masses is presented. The method is based on the estimation of the density of data points on MDiMs using Gaussian kernels followed by a curve fitting with an adapted version of the particle swarm optimization algorithm. The described recalibration procedure is examined on simulated as well as real mass spectrometric data. For the latter case, blood plasma samples were analyzed by Fourier transform ion cyclotron resonance mass spectrometry. Nevertheless, due to its inherent flexibility, the method can be easily extended to other low- and high-resolution platforms and/or sample types.

Metabolomic investigations in cerebrospinal fluid of Parkinson's disease.

The underlying mechanisms of Parkinson´s disease are not completely revealed. Especially, early diagnostic biomarkers are lacking. To characterize early pathophysiological events, research is focusing on metabolomics. In this case-control study we investigated the metabolic profile of 31 Parkinson´s disease-patients in comparison to 95 neurologically healthy controls. The investigation of metabolites in CSF was performed by a 12 Tesla SolariX Fourier transform-ion cyclotron resonance-mass spectrometer (FT-ICR-MS). Multivariate statistical analysis sorted the most important biomarkers in relation to their ability to differentiate Parkinson versus control. The affected metabolites, their connection and their conversion pathways are described by means of network analysis. The metabolic profiling by FT-ICR-MS in CSF yielded in a good group separation, giving insights into the disease mechanisms. A total number of 243 metabolites showed an affected intensity in Parkinson´s disease, whereas 15 of these metabolites seem to be the main biological contributors. The network analysis showed a connection to the tricarboxylic cycle (TCA cycle) and therefore to mitochondrial dysfunction and increased oxidative stress within mitochondria. The metabolomic analysis of CSF in Parkinson´s disease showed an association to pathways which are involved in lipid/ fatty acid metabolism, energy metabolism, glutathione metabolism and mitochondrial dysfunction.

Neonatal whisker trimming in WAG/Rij rat pups causes developmental delay, encourages maternal care and affects exploratory activity in adulthood.

WAG/Rij rats are genetically predisposed to absence epilepsy. Maternal behavior in WAG/Rij female rats is known to differ from that in non-epileptic females. We hypothesize that (1) mother's behavior may be changed as response to changes in pup's conditions; (2) sensory deprivation at the neonatal age affect learning and behavior in adulthood. All whiskers in WAG/Rij rat pups were trimmed daily during PN1-PN8. Maternal behavior was examined during the same period. It was found that in the control group, WAG/Rij females often demonstrated abnormally long (>1 min) repetitive purposeless stereotypical actions that were roughly classified as compulsive-like behavior. Mothers of the trimmed pups showed less compulsive-like behavior and more intensively interacted with pups and built better nests. Rat pups in the trimmed group had lower body weight on PN7-PN19 as compared to the control. In the trimmed group, maturation of motor skills and early behavioral patterns (i.e. walking, grooming, vertical activity, motor functions of forelimbs) showed 1-2 days delay in comparison to the control. At the age of 2-2.5 months, the locomotor activity in the trimmed rats differed from the control, but the level of anxiety was the same (the open field and the elevated plus maze). At the age of 6 months, the trimmed and control rats showed no differences in conditioned avoidance learning test, therefore, neonatal whisker trimming did not influence fear-based learning abilities in adulthood. It is hypothesized that an enhanced maternal care is capable to modulate development of brain functions in sensory deprived progeny.

How representative are dissolved organic matter (DOM) extracts? A comprehensive study of sorbent selectivity for DOM isolation.

Solid phase extraction (SPE) has become a widespread method for isolating dissolved organic matter (DOM) of diverse origin such as fresh and marine waters. This study investigated the DOM extraction selectivity of 24 commercially available SPE sorbents under identical conditions (pH = 2, methanol elution) on the example of Suwannee River (SR) water and North Sea (NS) water by using DOC analysis and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Proton nuclear magnetic resonance (1H NMR) spectroscopy was employed to assess leaching behavior, and HLB sorbent was found to leach substantially, among others. Variable DOC recoveries observed for SR DOM and NS DOM were primarily caused by the respective molecular composition, with subordinated and heterogeneous contributions of relative salinity. Scatter of average H/C and O/C elemental ratios and gross alignment in mass-edited H/C ratios according to five established coarse SPE characteristics was near identical for SR DOM and NS DOM. FTMS-based principal component analysis (PCA) provided essentially analogous alignment of SR DOM and NS DOM molecular compositions according to the five established groups of SPE classification, and corroborated the sorption-mechanism-based selectivity of DOM extraction in both cases. Evaluation of structural blanks and leaching of SPE cartridges requires NMR spectroscopy because FT-ICR mass spectrometry alone will not reveal inconspicuous displacements of continual bulk signatures caused by leaching of SPE resin constituents.

Oral versus intravenous iron replacement therapy distinctly alters the gut microbiota and metabolome in patients with IBD.

OBJECTIVE: Iron deficiency is a common complication in patients with IBD and oral iron therapy is suggested to exacerbate IBD symptoms. We performed an open-labelled clinical trial to compare the effects of per oral (PO) versus intravenous (IV) iron replacement therapy (IRT). DESIGN: The study population included patients with Crohn's disease (CD; N=31), UC (N=22) and control subjects with iron deficiency (non-inflamed, NI=19). After randomisation, participants received iron sulfate (PO) or iron sucrose (IV) over 3 months. Clinical parameters, faecal bacterial communities and metabolomes were assessed before and after intervention. RESULTS: Both PO and IV treatments ameliorated iron deficiency, but higher ferritin levels were observed with IV. Changes in disease activity were independent of iron treatment types. Faecal samples in IBD were characterised by marked interindividual differences, lower phylotype richness and proportions of Clostridiales. Metabolite analysis also showed separation of both UC and CD from control anaemic participants. Major shifts in bacterial diversity occurred in approximately half of all participants after IRT, but patients with CD were most susceptible. Despite individual-specific changes in phylotypes due to IRT, PO treatment was associated with decreased abundances of operational taxonomic units assigned to the species Faecalibacterium prausnitzii, Ruminococcus bromii, Dorea sp. and Collinsella aerofaciens. Clear IV-specific and PO-specific fingerprints were evident at the level of metabolomes, with changes affecting cholesterol-derived host substrates. CONCLUSIONS: Shifts in gut bacterial diversity and composition associated with iron treatment are pronounced in IBD participants. Despite similar clinical outcome, oral administration differentially affects bacterial phylotypes and faecal metabolites compared with IV therapy. TRIAL REGISTRATION NUMBER: clinicaltrial.gov (NCT01067547).