Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn's Disease.

OBJECTIVE: Epigenetic mechanisms, including DNA methylation (DNAm), have been proposed to play a key role in Crohn's disease (CD) pathogenesis. However, the specific cell types and pathways affected as well as their potential impact on disease phenotype and outcome remain unknown. We set out to investigate the role of intestinal epithelial DNAm in CD pathogenesis. DESIGN: We generated 312 intestinal epithelial organoids (IEOs) from mucosal biopsies of 168 patients with CD (n=72), UC (n=23) and healthy controls (n=73). We performed genome-wide molecular profiling including DNAm, bulk as well as single-cell RNA sequencing. Organoids were subjected to gene editing and the functional consequences of DNAm changes evaluated using an organoid-lymphocyte coculture and a nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 (NLRC5) dextran sulphate sodium (DSS) colitis knock-out mouse model. RESULTS: We identified highly stable, CD-associated loss of DNAm at major histocompatibility complex (MHC) class 1 loci including NLRC5 and cognate gene upregulation. Single-cell RNA sequencing of primary mucosal tissue and IEOs confirmed the role of NLRC5 as transcriptional transactivator in the intestinal epithelium. Increased mucosal MHC-I and NLRC5 expression in adult and paediatric patients with CD was validated in additional cohorts and the functional role of MHC-I highlighted by demonstrating a relative protection from DSS-mediated mucosal inflammation in NLRC5-deficient mice. MHC-I DNAm in IEOs showed a significant correlation with CD disease phenotype and outcomes. Application of machine learning approaches enabled the development of a disease prognostic epigenetic molecular signature. CONCLUSIONS: Our study has identified epigenetically regulated intestinal epithelial MHC-I as a novel mechanism in CD pathogenesis.

Resident gut microbiota community determines the efficacy of soluble fiber in reducing adiposity.

Consumption of dietary fiber has been linked to several health benefits. Among these, dietary fiber breakdown through the process of anaerobic fermentation by the colonic microbiota leads to the production of beneficial metabolites, mainly short-chain fatty acids (acetate, propionate, and butyrate), which have been implicated in reduced calorie intake. Nevertheless, the link between gut microbiota and obesity remains unclear. We investigated the effects of dietary fibers on food intake and body weight gain in two independent but similarly designed studies in rats. In the first study, the inclusion of 10% w/w pectin, fructooligosaccharides or beta-glucan (n = 10/group) in the diets each significantly reduced body weight gain ('responders') compared to the cellulose control whereas, in a closely matched, but not fully identical study (n = 8/group), no effect of dietary fiber on body weight ('non-responders') was observed. The aim of this work was to explore the basis of this differential response between the two similarly designed and comparable studies, with a focus on the potential role of the gut microbiota in the control of food intake and body weight.

Family Wellbeing and Sexual Health of Patients Receiving Treatment for Prostate Cancer.

Purpose: Prostate cancer and its treatment may affect patients' sexual function and social wellbeing. This study investigated the relationship between social/family wellbeing and sexual health in patients with prostate cancer. Additionally, the moderating effect of clinical characteristics on this relationship was also explored. Patients and Methods: This is a descriptive correlational study using baseline data of a longitudinal study enrolling 137 patients with prostate cancer. Sexual Function (SF) and Sexual Function Distress (SFD) data were collected using the Symptom Index questionnaire. Demographic data were obtained during study intake and clinical data were obtained from chart review. Bivariate correlation determined the correlations among continuous demographic/clinical data, social/family wellbeing, and sexual health. Moderated regression analysis determined the moderating effects of clinical characteristics on the relationship of social/family wellbeing and sexual health. Results: Moderate positive correlation was found between social/family wellbeing and SF, whereas a weak negative correlation was noted between social/family wellbeing and SFD. Depression was significantly correlated with social/family wellbeing and SFD. Both sexual health domains were significantly correlated with Gleason score. A significant difference was noted in the social/family wellbeing and both SF and SFD in participants receiving androgen deprivation therapy (ADT) compared to those not receiving ADT. Concomitant ADT use was the only clinical characteristic found to be a significant moderator of the relationship between social/family wellbeing and SFD, but none of the clinical characteristics was found to have a moderating effect on the relationship of social/family wellbeing and SF. Among patients who were not receiving ADT, high social/family wellbeing was associated with low SFD. Patients who were receiving ADT reported slightly higher SFD despite having higher social/family wellbeing. Conclusion: Ensuring sexual health in patients with prostate cancer requires a comprehensive approach to address factors contributing to sexual health such as side effects of treatment and family wellbeing.

A Roadmap for the Human Gut Cell Atlas.

The number of studies investigating the human gastrointestinal tract using various single-cell profiling methods has increased substantially in the past few years. Although this increase provides a unique opportunity for the generation of the first comprehensive Human Gut Cell Atlas (HGCA), there remains a range of major challenges ahead. Above all, the ultimate success will largely depend on a structured and coordinated approach that aligns global efforts undertaken by a large number of research groups. In this Roadmap, we discuss a comprehensive forward-thinking direction for the generation of the HGCA on behalf of the Gut Biological Network of the Human Cell Atlas. Based on the consensus opinion of experts from across the globe, we outline the main requirements for the first complete HGCA by summarizing existing data sets and highlighting anatomical regions and/or tissues with limited coverage. We provide recommendations for future studies and discuss key methodologies and the importance of integrating the healthy gut atlas with related diseases and gut organoids. Importantly, we critically overview the computational tools available and provide recommendations to overcome key challenges.

Erratum: Author Correction: A single-cell comparison of adult and fetal human epicardium defines the age-associated changes in epicardial activity.

[This corrects the article DOI: 10.1038/s44161-022-00183-w.].

Single-cell atlas of human liver development reveals pathways directing hepatic cell fates.

The liver has been studied extensively due to the broad number of diseases affecting its vital functions. However, therapeutic advances have been hampered by the lack of knowledge concerning human hepatic development. Here, we addressed this limitation by describing the developmental trajectories of different cell types that make up the human liver at single-cell resolution. These transcriptomic analyses revealed that sequential cell-to-cell interactions direct functional maturation of hepatocytes, with non-parenchymal cells playing essential roles during organogenesis. We utilized this information to derive bipotential hepatoblast organoids and then exploited this model system to validate the importance of signalling pathways in hepatocyte and cholangiocyte specification. Further insights into hepatic maturation also enabled the identification of stage-specific transcription factors to improve the functionality of hepatocyte-like cells generated from human pluripotent stem cells. Thus, our study establishes a platform to investigate the basic mechanisms directing human liver development and to produce cell types for clinical applications.

Neural mass model-based study of frontal-temporal theta oscillations in human subjects during the performance of a cognitive control task.

Cognitive control, the ability to rapidly shift one's attention and behavioral strategy in response to environmental changes, is often compromised across psychiatric disorders. One of the well-validated behavioral paradigms for tapping into the cognitive control circuits is a cognitive interference task, where subjects must suppress a natural response to follow a less intuitive rule. Slower response times on these tasks indicate difficulty exerting control to overcome response conflict. Conflict evokes robust electrophysiological signatures, such as theta (4-8 Hz) oscillations in the prefrontal cortex (PFC). However, the underlying neural mechanisms of conflict-evoked theta oscillations in the PFC are not clear. The objective of this work is to use a neural mass model (NMM) to find feasible cortical networks generating theta oscillations during conflict processing in human subjects. We used intracranial EEG (iEEG) recorded from dorsolateral PFC (dIPFC) and lateral temporal lobe (LTL) of human subjects with intractable epilepsy undergoing invasive monitoring, while they performed a multi-source interference task (MSIT). We used a dynamic causal modeling (DCM) framework to simulate dIPFC-LTL theta using a Jansen-Rit NMM. We found significant evidence for an LTL input into the dlPFC during the initial 500 ms of conflict processing compared to a bidirectional connection between the dlPFC and LTL. We conclude that a neural mass modeling framework can be used to elucidate candidate mechanisms of neural oscillations underlying conflict resolution in human subjects. Clinical Relevance- This can be used to find feasible target mechanisms for designing therapy in patients with compromised cognitive control. This framework can also be expanded to serve as an in-silico test bed for designing and testing neuromodulatory interventions such as electrical stimulation for improving cognitive control in mood/anxiety disorders.

A single-cell comparison of adult and fetal human epicardium defines the age-associated changes in epicardial activity.

Re-activating quiescent adult epicardium represents a potential therapeutic approach for human cardiac regeneration. However, the exact molecular differences between inactive adult and active fetal epicardium are not known. In this study, we combined fetal and adult human hearts using single-cell and single-nuclei RNA sequencing and compared epicardial cells from both stages. We found that a migratory fibroblast-like epicardial population only in the fetal heart and fetal epicardium expressed angiogenic gene programs, whereas the adult epicardium was solely mesothelial and immune responsive. Furthermore, we predicted that adult hearts may still receive fetal epicardial paracrine communication, including WNT signaling with endocardium, reinforcing the validity of regenerative strategies that administer or reactivate epicardial cells in situ. Finally, we explained graft efficacy of our human embryonic stem-cell-derived epicardium model by noting its similarity to human fetal epicardium. Overall, our study defines epicardial programs of regenerative angiogenesis absent in adult hearts, contextualizes animal studies and defines epicardial states required for effective human heart regeneration.

Similar zooplankton responses to low pH and calcium may impair long-term recovery from acidification.

Throughout much of the 20th century, unprecedented industrial emissions have led to widespread acidification of regions in North America and Europe and, as lake water pH dropped, aquatic ecosystems have experienced dramatic declines in biodiversity. International emission-control agreements have led to sweeping increases in lake pH, however acid-structured zooplankton communities still persist in many lakes. Concomitantly, calcium concentrations have been declining as a legacy of acidification and are approaching or have reached concentrations that could represent a barrier to the re-establishment of zooplankton communities similar to those in non-acidified or circumneutral reference lakes. To understand how declining calcium may influence the re-establishment of zooplankton in acid-damaged lakes we manipulated calcium and pH using a factorial in-lake mesocosm experiment and assessed their individual and combined effects on a regionally diverse zooplankton assemblage. We found that the impacts of low calcium on zooplankton species were similar to those of acidification and, consequently, may prevent the recovery of acid-structured communities. Abundance of the larger bodied and acid-sensitive Daphnia pulex/pulicaria increased in high pH treatments, albeit nonsignificantly yet, by the end of our experiment, only two individuals were sampled among our 10 low calcium enclosures. In contrast, small acid-tolerant cladocerans, such as Daphnia catawba, Daphnia ambigua, and eubosminids maintained significantly higher abundances in low calcium treatments relative to all other treatment combinations. Although we did not detect an effect of calcium on mean body size, the disproportionately high abundance of small cladocerans in low calcium treatments resulted in low calcium communities with higher overall abundance and lower cladoceran evenness. Our results, along with a landscape comparison demonstrating parallel changes in zooplankton relative abundance from 34 historically acidified lakes, suggests that declining calcium will be an important, on-going factor that may limit the recovery of zooplankton, despite regional improvements in lake pH.

Modulation of pulsatile GnRH dynamics across the ovarian cycle via changes in the network excitability and basal activity of the arcuate kisspeptin network.

Pulsatile GnRH release is essential for normal reproductive function. Kisspeptin secreting neurons found in the arcuate nucleus, known as KNDy neurons for co-expressing neurokinin B, and dynorphin, drive pulsatile GnRH release. Furthermore, gonadal steroids regulate GnRH pulsatile dynamics across the ovarian cycle by altering KNDy neurons' signalling properties. However, the precise mechanism of regulation remains mostly unknown. To better understand these mechanisms, we start by perturbing the KNDy system at different stages of the estrous cycle using optogenetics. We find that optogenetic stimulation of KNDy neurons stimulates pulsatile GnRH/LH secretion in estrous mice but inhibits it in diestrous mice. These in vivo results in combination with mathematical modelling suggest that the transition between estrus and diestrus is underpinned by well-orchestrated changes in neuropeptide signalling and in the excitability of the KNDy population controlled via glutamate signalling. Guided by model predictions, we show that blocking glutamate signalling in diestrous animals inhibits LH pulses, and that optic stimulation of the KNDy population mitigates this inhibition. In estrous mice, disruption of glutamate signalling inhibits pulses generated via sustained low-frequency optic stimulation of the KNDy population, supporting the idea that the level of network excitability is critical for pulse generation. Our results reconcile previous puzzling findings regarding the estradiol-dependent effect that several neuromodulators have on the GnRH pulse generator dynamics. Therefore, we anticipate our model to be a cornerstone for a more quantitative understanding of the pathways via which gonadal steroids regulate GnRH pulse generator dynamics. Finally, our results could inform useful repurposing of drugs targeting the glutamate system in reproductive therapy.

Magneto-Activation and Magnetic Resonance Imaging of Natural Killer Cells Labeled with Magnetic Nanocomplexes for the Treatment of Solid Tumors.

Natural killer (NK) cell-based immunotherapy has been considered a promising cell-based cancer treatment strategy with low side effects for early tumors and metastasis. However, the therapeutic efficacy is generally low in established solid tumors. Ex vivo activation of NK cells with exogenous cytokines is often essential but ineffective to generate high doses of functional NK cells for cancer treatment. Image-guided local delivery of NK cells is also suggested for the therapy. However, there is a lack of noninvasive tools for monitoring NK cells. Herein, magnetic nanocomplexes are fabricated with clinically available materials (hyaluronic acid, protamine, and ferumoxytol; HAPF) for labeling NK cells. The prepared HAPF-nanocomplexes effectively attach to the NK cells (HAPF-NK). An exogenous magnetic field application effectively achieves magneto-activation of NK cells, promoting the generation and secretion of lytic granules of NK cells. The magneto-activated HAPF-NK cells also allow an MR image-guided NK cell therapy to treat hepatocellular carcinoma (HCC) solid tumors via transcatheter intra-arterial infusion. Suppressed tumor growth after the treatment of IA infused magneto-activated NK cells demonstrated a potential enhanced therapeutic efficacy of image guided local delivery of magneto-activated HAPF-NK cells. Given the potential challenges of NK cell cancer immunotherapy against established solid tumors, the effective NK cell labeling with HAPF, magneto-activation, and MRI contrast effect of NK cells will be beneficial to enhance the NK cell-therapeutic efficacy in various cancers.

Obtaining purified human intestinal epithelia for single-cell analysis and organoid culture.

Here, we describe protocols for the preparation and dissociation of human fetal and pediatric intestinal tissue to a high-viability epithelial single-cell suspension. This epithelium-enriched single-cell suspension can then be used to generate single-cell RNA sequencing data as well as to create human intestinal organoids from both the fetal and pediatric intestine. Finally, this protocol details the dissociation of the intestinal organoids for use in single-cell analysis or passaging of organoids. For complete details on the use and execution of this protocol, please refer to Elmentaite et al. (2020).

Early mobilization vs delayed mobilisation following the use of a volar locking plate with non-vascularized bone graft in scaphoid non-union. A multicentred randomised controlled-trial.

PURPOSE: This randomized clinical trial investigated the potential for early mobilization of the wrist following open reduction and internal fixation (ORIF) with a scaphoid specific volar locking plate and non-vascularized bone graft for scaphoid non-union. METHODS: 16 patients with scaphoid non-union underwent internal fixation with a scaphoid-specific volar locking plate and iliac crest bone graft and were randomized to one of two treatment arms (A) The control were immobilized in a below elbow cast for 6 weeks (n = 9) and (B) The experimental arm were mobilized early with a removable wrist splint (n = 7). Outcomes were measured preoperatively, and at 3 months post operatively. These included the primary outcome of union, and secondary outcomes of grip strength and patient reported outcomes of disabilities of arm shoulder and hand (DASH) and patient reported wrist evaluation (PRWE). Discrete variables were analyzed using the chi squared test while continuous variables used the students t-test. RESULTS: The experimental (early mobilization) group developed metalware complications resulting in the early termination of the study. No significant difference in the demographic characteristics of age, gender, time to surgery, smoking status and handedness was found between groups. A significant difference was found in BMI, with significantly higher proportion of obese patients (p = 0.05) in the experimental group. There was no significant difference in the primary outcome measure of the rate of union between groups. The secondary outcomes of grip strength, Dash and PRWE also showed no significant difference between the immobilized and mobilized groups. CONCLUSION: We recommend immobilization following scaphoid non-union ORIF using a volar locking plate due to high complication rates in our cohort with early mobilization.Type of study/level of evidence: Therapeutic 2.Trial registration.Australian New Zealand Clinical Trials Registry (ACTRN12614001050640). Date of registration, 02/10/2014.

Regional Differences in Human Biliary Tissues and Corresponding In Vitro-Derived Organoids.

BACKGROUND AND AIMS: Organoids provide a powerful system to study epithelia in vitro. Recently, this approach was applied successfully to the biliary tree, a series of ductular tissues responsible for the drainage of bile and pancreatic secretions. More precisely, organoids have been derived from ductal tissue located outside (extrahepatic bile ducts; EHBDs) or inside the liver (intrahepatic bile ducts; IHBDs). These organoids share many characteristics, including expression of cholangiocyte markers such as keratin (KRT) 19. However, the relationship between these organoids and their tissues of origin, and to each other, is largely unknown. APPROACH AND RESULTS: Organoids were derived from human gallbladder, common bile duct, pancreatic duct, and IHBDs using culture conditions promoting WNT signaling. The resulting IHBD and EHBD organoids expressed stem/progenitor markers leucine-rich repeat-containing G-protein-coupled receptor 5/prominin 1 and ductal markers KRT19/KRT7. However, RNA sequencing revealed that organoids conserve only a limited number of regional-specific markers corresponding to their location of origin. Of particular interest, down-regulation of biliary markers and up-regulation of cell-cycle genes were observed in organoids. IHBD and EHBD organoids diverged in their response to WNT signaling, and only IHBDs were able to express a low level of hepatocyte markers under differentiation conditions. CONCLUSIONS: Taken together, our results demonstrate that differences exist not only between extrahepatic biliary organoids and their tissue of origin, but also between IHBD and EHBD organoids. This information may help to understand the tissue specificity of cholangiopathies and also to identify targets for therapeutic development.

Transcription and DNA Methylation Patterns of Blood-Derived CD8+ T Cells Are Associated With Age and Inflammatory Bowel Disease But Do Not Predict Prognosis.

BACKGROUND & AIMS: Gene expression patterns of CD8+ T cells have been reported to correlate with clinical outcomes of adults with inflammatory bowel diseases (IBD). We aimed to validate these findings in independent patient cohorts. METHODS: We obtained peripheral blood samples from 112 children with a new diagnosis of IBD (71 with Crohn's disease and 41 with ulcerative colitis) and 19 children without IBD (controls) and recorded medical information on disease activity and outcomes. CD8+ T cells were isolated from blood samples by magnetic bead sorting at the point of diagnosis and during the course of disease. Genome-wide transcription (n = 192) and DNA methylation (n = 66) profiles were generated using Affymetrix and Illumina arrays, respectively. Publicly available transcriptomes and DNA methylomes of CD8+ T cells from 3 adult patient cohorts with and without IBD were included in data analyses. RESULTS: Previously reported CD8+ T-cell prognostic expression and exhaustion signatures were only found in the original adult IBD patient cohort. These signatures could not be detected in either a pediatric or a second adult IBD cohort. In contrast, an association between CD8+ T-cell gene expression with age and sex was detected across all 3 cohorts. CD8+ gene transcription was clearly associated with IBD in the 2 cohorts that included non-IBD controls. Lastly, DNA methylation profiles of CD8+ T cells from children with Crohn's disease correlated with age but not with disease outcome. CONCLUSIONS: We were unable to validate previously reported findings of an association between CD8+ T-cell gene transcription and disease outcome in IBD. Our findings reveal the challenges of developing prognostic biomarkers for patients with IBD and the importance of their validation in large, independent cohorts before clinical application.

Single-Cell Sequencing of Developing Human Gut Reveals Transcriptional Links to Childhood Crohn's Disease.

Human gut development requires the orchestrated interaction of differentiating cell types. Here, we generate an in-depth single-cell map of the developing human intestine at 6-10 weeks post-conception. Our analysis reveals the transcriptional profile of cycling epithelial precursor cells; distinct from LGR5-expressing cells. We propose that these cells may contribute to differentiated cell subsets via the generation of LGR5-expressing stem cells and receive signals from surrounding mesenchymal cells. Furthermore, we draw parallels between the transcriptomes of ex vivo tissues and in vitro fetal organoids, revealing the maturation of organoid cultures in a dish. Lastly, we compare scRNA-seq profiles from pediatric Crohn's disease epithelium alongside matched healthy controls to reveal disease-associated changes in the epithelial composition. Contrasting these with the fetal profiles reveals the re-activation of fetal transcription factors in Crohn's disease. Our study provides a resource available at www.gutcellatlas.org, and underscores the importance of unraveling fetal development in understanding disease.

Whole-heart, ungated, free-breathing, cardiac-phase-resolved myocardial perfusion MRI by using Continuous Radial Interleaved simultaneous Multi-slice acquisitions at sPoiled steady-state (CRIMP).

PURPOSE: To develop a whole-heart, free-breathing, non-electrocardiograph (ECG)-gated, cardiac-phase-resolved myocardial perfusion MRI framework (CRIMP; Continuous Radial Interleaved simultaneous Multi-slice acquisitions at sPoiled steady-state) and test its quantification feasibility. METHODS: CRIMP used interleaved radial simultaneous multi-slice (SMS) slice groups to cover the whole heart in 9 or 12 short-axis slices. The sequence continuously acquired data without magnetization preparation, ECG gating or breath-holding, and captured multiple cardiac phases. Images were reconstructed by a motion-compensated patch-based locally low-rank reconstruction. Bloch simulations were performed to study the signal-to-noise ratio/contrast-to-noise ratio (SNR/CNR) for CRIMP and to study the steady-state signal under motion. Seven patients were scanned with CRIMP at stress and rest to develop the sequence. One human and two dogs were scanned at rest with a dual-bolus method to test the quantification feasibility of CRIMP. The dual-bolus scans were performed using both CRIMP and an ungated radial SMS saturation recovery (SMS-SR) sequence with injection dose = 0.075 mmol/kg to compare the sequences in terms of SNR, cardiac phase resolution and quantitative myocardial blood flow (MBF). RESULTS: Perfusion images with multiple cardiac phases in all image slices with a temporal resolution of 72 ms/frame were obtained. Simulations and in-vivo acquisitions showed CRIMP kept the inner slices in steady-state regardless of motion. CRIMP outperformed SMS-SR in slice coverage (9 over 6), SNR (mean 20% improvement), and provided cardiac phase resolution. CRIMP and SMS-SR sequences provided comparable MBF values (rest systolic CRIMP = 0.58 ± 0.07, SMS-SR = 0.61 ± 0.16). CONCLUSION: CRIMP allows for whole-heart, cardiac-phase-resolved myocardial perfusion images without ECG-gating or breath-holding. The sequence can provide MBF if an accurate arterial input function is obtained separately.