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Cholangiopathies are poorly understood disorders with no effective therapy. The extrahepatic biliary tree phenotype is less studied compared to the intrahepatic biliary injury in both human disease and Mdr2 -/- mice, the established cholestatic mouse model. This study aimed to characterize the extra hepatic biliary tree of Mdr2 -/- mice at various ages and to determine if injury can be repaired with the antioxidant and glutathione precursor N-acetyl-L-Cysteine treatment (NAC). We characterized extra hepatic bile ducts (EHBD)s at various ages from 2 to 40 weeks old FVB/N and Mdr2 -/- mice. We examined the therapeutic potential of local NAC ex vivo using EHBD explants at early and late stages of injury; and systematic therapy by in vivo oral administration for 3 weeks. EHBD and liver sections were assessed by histology and immunofluorescent stains. Serum liver enzyme activities were analyzed, and liver spatial protein expression analysis was performed. Mdr2 -/- mice developed progressive EHBD injury, similar to extrahepatic PSC. NAC treatment of ex vivo EHBD explants led to improved duct morphology. In vivo, oral administration of NAC improved liver fibrosis, and decreased liver enzyme activities. Spatial protein analysis revealed cell-type specific differential response to NAC, collectively indicating a transition from pro-apoptotic into proliferative state. NAC treatment should be further investigated as a potential therapeutic option for human cholangiopathies., (© 2024. The Author(s).)

Background: Elevated spleen stiffness may be seen in patients with portal hypertension due to cirrhosis. In patients with Fontan physiology, elevated liver stiffness has been shown to correlate poorly with liver fibrosis. It is unknown whether spleen stiffness may instead serve as a surrogate marker of liver fibrosis in these patients., Objective: To compare spleen stiffness determined by shear wave elastography (SWE) with histological findings of an ultrasound-guided liver biopsy in patients who had undergone Fontan palliation as a potential surrogate for Fontan-associated liver disease., Materials and Methods: This was an IRB-approved single-center, retrospective study. Patients with Fontan palliation who had undergone both a spleen SWE study and a percutaneous liver biopsy between 2016 and 2020 were included. Biopsy, performed during cardiac catheterization, within 3 months of the SWE was required for inclusion. Using Kruskal-Wallis tests, spleen stiffness was compared with three liver biopsy scoring methods: Ishak, METAVIR, and congestive hepatic fibrosis score (CHFS). When available, Pearson's correlation was also used to compare collagen deposition determined using Sirius Red stain (%SR) with SWE values. A P-value < 0.05 was considered statistically significant., Results: Twenty-two patients (15 males) were included in the study, with a median age of 17 years (IQR is 14.8-20.5 years; age range: 7 years to 30.2 years). The median spleen stiffness was 2.94 m/s (IQR: 2.57-3.61 m/s; range: 1.48-4.27 m/s). The median Fontan pressure was 11 mm Hg (IQR: 10-13.3 mm Hg; range: 7-19 mm Hg) obtained within a median of 10 days (IQR: 1-41 days) of SWE. Splenic stiffness did not correlate with the extent of fibrosis determined by histology (all P > 0.05). There was also no statistically significant correlation between the %SR staining and SWE-determined spleen stiffness (Pearson's correlation of 0.165, P = 0.59, n = 13)., Conclusions: In this preliminary study, SWE spleen stiffness values did not correlate with biopsy-determined scoring of liver fibrosis in patients with Fontan physiology., Competing Interests: Declarations. Conflicts of interest: None Publication: This manuscript is not under consideration for publication elsewhere, except for the abstract that was accepted for a Scientific Presentation during the SPR [Society for Pediatric Radiology] 2023 Annual Meeting & Postgraduate Course., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background & Aims: Hirschsprung's disease is defined by the absence of the enteric nervous system (ENS) from the distal bowel. Primary treatment is "pull-through" surgery to remove bowel that lacks ENS, with reanastomosis of "normal" bowel near the anal verge. Problems after pull-through are common, and some may be due to retained hypoganglionic bowel (ie, low ENS density). Testing this hypothesis has been difficult because counting enteric neurons in tissue sections is unreliable, even for experts. Tissue clearing and 3-dimensional imaging provide better data about ENS structure than sectioning., Methods: Regions from 11 human colons and 1 ileal specimen resected during Hirschsprung's disease pull-through surgery were cleared, stained with antibodies to visualize the ENS, and imaged by confocal microscopy. Control distal colon from people with no known bowel problems were similarly cleared, stained, and imaged., Results: Quantitative analyses of human colon, ranging from 3 days to 60 years old, suggest age-dependent changes in the myenteric plexus area, ENS ganglion area, percentage of myenteric plexus occupied by ganglia, neurons/mm 2 , and neuron Feret's diameter. Neuron counting using 3-dimensional images was highly reproducible. High ENS density in neonatal colon allowed reliable neuron counts using 500-μm 2  × 500-μm 2 regions (36-fold smaller than in adults). Hirschsprung's samples varied 8-fold in proximal margin enteric neuron density and had diverse ENS architecture in resected bowel., Conclusions: Tissue clearing and 3-dimensional imaging provide more reliable information about ENS structure than tissue sections. ENS structure changes during childhood. Three-dimensional ENS anatomy may provide new insight into human bowel motility disorders, including Hirschsprung's disease., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

We present the first case of a dupilumab-induced hyperinflammatory state in the setting of underlying eosinophilic esophagitis characterized by multisystem granulomatous inflammation. Although clinical trial data and subsequent real-world experience support dupilumab as a highly effective therapy for eosinophilic esophagitis, close monitoring for development of adverse symptoms following initiation remains paramount., Competing Interests: Disclosure of potential conflict of interest: A. B. Muir receives consulting fees from Bristol-Meyers Squib, Nexstone Immunology, and 10.13039/100009857Regeneron, as well as research funding from 10.13039/100030947Allakos and Morphic. The rest of the authors declare that they have no relevant conflicts of interest., (© 2024 The Authors.)

Background: Pediatric granular cell tumors (GCT) involving the gastrointestinal tract (GIT) are rare with limited case report/series reported to date., Methods: Multicenter retrospective study of pediatric GIT GCT., Results: A total of 10 cases were included in the study with a median age of 13.5 years (range: 7-18 years) and were predominantly female patients (60%). In half of the patients no significant medical history was present with the remaining 5 having Crohn disease (10%), eosinophilic esophagitis (EoE) (10%), Crohn disease and EoE (10%), growth hormone deficiency (10%), and aplasia cutis congenita (10%). The GCT median size was 1.3 cm (range: 1-1.6 cm) and were more commonly located in the esophagus (70%) followed by the stomach (20%) and rectum (10%). Most of the cases showed round/polygonal tumor cells with abundant granular cytoplasm, and none of the cases had nuclear atypia, increased mitotic activity, or tumor cell necrosis. None of our cases received specific therapy for GCT other than clinical follow-up, and none of the patients had evidence of local recurrence or metastatic disease., Conclusion: We present our multicenter experience with GIT GCT, all cases had a benign course. Interestingly, 4 of the esophageal GCT cases (including 2 patients with EoE) showed an eosinophil-rich esophagitis in the underlying mucosa., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The Fontan operation is the current standard of care for single-ventricle congenital heart disease. Individuals with a Fontan circulation (FC) exhibit central venous hypertension and face life-threatening complications of hepatic fibrosis, known as Fontan-associated liver disease (FALD). The fundamental biology and mechanisms of FALD are little understood. Here, we generated a transcriptomic and epigenomic atlas of human FALD at single-cell resolution using multiomic snRNA-ATAC-seq. We found profound cell type-specific transcriptomic and epigenomic changes in FC livers. Central hepatocytes (cHep) exhibited the most substantial changes, featuring profound metabolic reprogramming. These cHep changes preceded substantial activation of hepatic stellate cells and liver fibrosis, suggesting cHep as a potential first "responder" in the pathogenesis of FALD. We also identified a network of ligand-receptor pairs that transmit signals from cHep to hepatic stellate cells, which may promote their activation and liver fibrosis. We further experimentally demonstrated that activins A and B promote fibrotic activation in vitro and identified mechanisms of activin A's transcriptional activation in FALD. Together, our single-cell transcriptomic and epigenomic atlas revealed mechanistic insights into the pathogenesis of FALD and may aid identification of potential therapeutic targets.

Zebrafish ( Danio rerio ) is a widely used vertebrate animal for modeling genetic diseases by targeted editing strategies followed by gross phenotypic and biomarker characterization. While larval transparency permits microscopic detection of anatomical defects, histological adult screening for organ-level defects remains invasive, tedious, inefficient, and subject to technical artifact. Here, we describe a noninvasive magnetic resonance imaging (MRI) approach to systematically screen adult zebrafish for anatomical growth defects. An anatomical atlas of wild-type (WT) zebrafish at 5-31 months post-fertilization was created by ex vivo MRI with a 9.4 T magnet. Volumetric growth over time was measured of animals and major organs, including the brain, spinal cord, heart, eyes, optic nerve, ear, liver, kidneys, and swim bladder. Subsequently, surf1 -/- , fbxl4 -/- , and opa1 +/- mitochondrial disease mutant adult zebrafish were quantitatively studied to compare organ volumes with age-matched WT zebrafish. Results demonstrated that MRI enabled noninvasive, high-resolution, rapid screening of mutant adult zebrafish for overall and organ-specific growth abnormalities. Detailed volumetric analyses of three mitochondrial disease mutants delineated specific organ differences, including significantly increased brain growth in surf1 -/- and opa1 +/- , and marginally significant decreased heart and spinal cord volumes in surf1 -/- mutants. This is interesting as we know neurological involvement can be seen in SURF1 - /- patients with ataxia, dystonia, and lesions in basal ganglia, as well as in OPA1 +/- patients with spasticity, ataxia, and hyperreflexia indicative of neuropathology. Similarly, cardiomyopathy is a known sequelae of cardiac pathology in patients with SURF1 - /- -related disease. Future studies will define MRI signaling patterns of organ dysfunction to further delineate specific pathology.

Intestinal epithelial transit-amplifying cells are essential stem progenitors required for intestinal homeostasis, but their rapid proliferation renders them vulnerable to DNA damage from radiation and chemotherapy. Despite these cells' critical roles in intestinal homeostasis and disease, few studies have described genes that are essential to transit-amplifying cell function. We report that RNA methyltransferase-like 3 (METTL3) is required for survival of transit-amplifying cells in the murine small intestine. Transit-amplifying cell death after METTL3 deletion was associated with crypt and villus atrophy, loss of absorptive enterocytes, and uniform wasting and death in METTL3-depleted mice. Sequencing of polysome-bound and methylated RNAs in enteroids and in vivo demonstrated decreased translation of hundreds of methylated transcripts after METTL3 deletion, particularly transcripts involved in growth factor signal transduction such as Kras. Further investigation verified a relationship between METTL3 and Kras methylation and protein levels in vivo. Our study identifies METTL3 as an essential factor supporting the homeostasis of small intestinal tissue via direct maintenance of transit-amplifying cell survival. We highlight the crucial role of RNA modifications in regulating growth factor signaling in the intestine with important implications for both homeostatic tissue renewal and epithelial regeneration.

Biliary atresia is a fibroinflammatory neonatal disease with no effective therapies. A subset of cases (10-20%) is associated with laterality defects - labeled biliary atresia splenic malformation (BASM) syndrome. Recently, whole-exome sequencing of patients with BASM identified deleterious variants in PKD1L1. PKD1L1 is involved in left-right axis determination; however, its role in cholangiocytes is unknown. We generated the pkd1l1hsc117 allele using CRISPR/Cas9 mutagenesis in zebrafish to determine the role of Pkd1l1 in biliary development and function. Wild-type and mutant larvae were assessed for laterality defects, biliary function and biliary tree architecture at 5 days post fertilization. pkd1l1hsc117 mutant larvae exhibited early left-right patterning defects. The gallbladder was positioned on the left in 47% of mutants compared to 4% of wild-type larvae. Accumulation of PED6 in the gallbladder, an indicator of hepatobiliary function, was significantly reduced in pkd1l1hsc117 mutants (46%) compared to wild-type larvae (4%). pkd1l1hsc117 larvae exhibited fewer biliary epithelial cells and reduced density of the intrahepatic biliary network compared to those in wild-type larvae. These data highlight the essential role of pkd1l1 in normal development and function of the zebrafish biliary system, supporting a role for this gene as a cause of BASM., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

The utilization of an expanded genetic code and in vivo unnatural amino acid crosslinking has grown significantly in the past decade, proving to be a reliable system for the examination of protein-protein interactions. Perhaps the most utilized amino acid crosslinker, p -benzoyl-(l)-phenylalanine (pBPA), has delivered a vast compendium of structural and mechanistic data, placing it firmly in the upper echelons of protein analytical techniques. pBPA contains a benzophenone group that is activated with low energy radiation (~365 nm), initiating a diradical state that can lead to hydrogen abstraction and radical recombination in the form of a covalent bond to a neighboring protein. Importantly, the expanded genetic code system provides for site-specific encoding of the crosslinker, yielding spatial control for protein surface mapping capabilities. Paired with UV-activation, this process offers a practical means for spatiotemporal understanding of protein-protein dynamics in the living cell. The chromatin field has benefitted particularly well from this technique, providing detailed mapping and mechanistic insight for numerous chromatin-related pathways. We provide here a brief history of unnatural amino acid crosslinking in chromatin studies and outlooks into future applications of the system for increased spatiotemporal resolution in chromatin related research.

Intestinal epithelial transit amplifying cells are essential stem progenitors required for intestinal homeostasis, but their rapid proliferation renders them vulnerable to DNA damage from radiation and chemotherapy. Despite their critical roles in intestinal homeostasis and disease, few studies have described genes that are essential to transit amplifying cell function. We report that the RNA methyltransferase, METTL3, is required for survival of transit amplifying cells in the murine small intestine. Transit amplifying cell death after METTL3 deletion was associated with crypt and villus atrophy, loss of absorptive enterocytes, and uniform wasting and death in METTL3-depleted mice. Ribosome profiling and sequencing of methylated RNAs in enteroids and in vivo demonstrated decreased translation of hundreds of unique methylated transcripts after METTL3 deletion, particularly transcripts involved in growth factor signal transduction such as Kras . Further investigation confirmed a novel relationship between METTL3 and Kras methylation and protein levels in vivo . Our study identifies METTL3 as an essential factor supporting the homeostasis of small intestinal tissue via direct maintenance of transit amplifying cell survival. We highlight the crucial role of RNA modifications in regulating growth factor signaling in the intestine, with important implications for both homeostatic tissue renewal and epithelial regeneration., Competing Interests: Conflict-of-interest statement Premal Shah is a member of the Scientific Advisory Board of Trestle Biosciences and is Director at Ananke Therapeutics. All other authors declare they have no competing interests.

Introduction: Inflammation in eosinophilic esophagitis (EoE) often leads to esophageal strictures. Evaluating esophageal narrowing is clinically challenging. We evaluated esophageal distensibility as related to disease activity, fibrosis, and dysphagia., Methods: Adult patients with and without EoE underwent endoscopy and distensibility measurements. Histology, distensibility, and symptoms were analyzed., Results: Patients with EoE had significantly lower distensibilities than controls. We found a cohort with esophageal diameter under 15 mm despite lack of dysphagia., Discussion: This study raises concern that current assessments of fibrostenosis are suboptimal. We describe a cohort with unrecognized slender esophagus that were identified through impedance planimetry measurements. This tool provides additional information beyond symptomatic, histologic, and endoscopic assessments., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

The RNA-binding protein Pcbp2 is widely expressed in the innate and adaptive immune systems and is essential for mouse development. To determine whether Pcbp2 is required for CD4 + T cell development and function, we derived mice with conditional Pcbp2 deletion in CD4 + T cells and assessed their overall phenotype and proliferative responses to activating stimuli. We found that Pcbp2 is essential for T conventional cell (Tconv) proliferation, working through regulation of co-stimulatory signaling. Pcbp2 deficiency in the CD4 + lineage did not impact Treg abundance in vivo or function in vitro . In addition, our data demonstrate a clear association between Pcbp2 control of Runx1 exon 6 splicing in CD4 + T cells and a specific role for Pcbp2 in the maintenance of peripheral CD4 + lymphocyte population size. Last, we show that Pcbp2 function is required for optimal in vivo Tconv cell activation in a T cell adoptive transfer colitis model system., Competing Interests: We have no conflicts of interest to declare., (© 2022 The Author(s).)

We present a case of solid-tubulocystic variant of intrahepatic cholangiocarcinoma (also called cholangioblastic variant of intrahepatic cholangiocarcinoma) in a 15-year-old girl, the youngest patient reported to date. The tumor was located in the left lobe of the liver, predominantly solid with cystic areas, and measured 16 cm in greatest dimension. Microscopic examination showed 2 major histologic patterns: a mixed pattern with solid, tubulocystic, macrocystic, trabecular, and nested growth, diffuse cytokeratin 7/19 and weak neuroendocrine immunoreactivity, and low Ki-67 index; and a more compact, macrotrabecular/gyriform pattern with focal CK7/19, stronger neuroendocrine reactivity, and higher Ki-67 index. Inhibin immunoreactivity was diffuse throughout both patterns. Treatment included tumor resection with negative margins and 8 cycles of capecitabine chemotherapy; the patient is alive with no evidence of tumor 2.5 years after resection. Although molecular characterization of the tumor at the time of resection was unrevealing, a recent study has identified a novel NIPBL-NACC1 fusion transcript in this tumor type, which we have confirmed in this case. This case expands the reported age range of this rare tumor type and confirms a recently-reported diagnostic genomic alteration. Awareness of this rare entity affecting pediatric patients is crucial to avoid confusion with similar-appearing neoplasms.

Here, we introduce a facile, scalable engineering approach to enable long-term development and maturation of organoids. We have redesigned the configuration of conventional organoid culture to develop a platform that converts single injections of stem cell suspensions to radial arrays of organoids that can be maintained for extended periods without the need for passaging. Using this system, we demonstrate accelerated production of intestinal organoids with significantly enhanced structural and functional maturity, and their continuous development for over 4 weeks. Furthermore, we present a patient-derived organoid model of inflammatory bowel disease (IBD) and its interrogation using single-cell RNA sequencing to demonstrate its ability to reproduce key pathological features of IBD. Finally, we describe the extension of our approach to engineer vascularized, perfusable human enteroids, which can be used to model innate immune responses in IBD. This work provides an immediately deployable platform technology toward engineering more realistic organ-like structures in a dish., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Aim: To compare targeted and global liver stiffness measured by magnetic resonance elastography (MRE) with liver biopsy in patients who have undergone the Fontan procedure, and to assess the relationship between liver stiffness and fibrosis stage., Materials and Methods: Targeted and global liver stiffness was compared with a quantification of liver fibrosis measured by percentage of Sirius Red (%SR) staining of biopsy samples. MRE values were compared with three other biopsy-scoring methods: Ishak, Scheuer/Ludwig-Batts/Metavir, and congestive hepatic fibrosis score (CHFS). Additionally, in patients who had two or more MRE studies, global liver stiffness was compared for longitudinal assessment., Results: Thirty-four patients were included in the study, with a mean age of 16.2 years. There was no statistically significant correlation between MRE-derived liver stiffness and Ishak score, Metavir score, %SR staining, and CHFS score. Twenty patients had multiple MRE studies, with a mean age of 16.5 years, and these showed a statistically significant increase in mean liver stiffness from 3.72 to 4.68 (26% increase) within an average period of 24 months., Conclusions: The lack of correlation of liver stiffness with fibrosis stage observed in this study indicates that the effects of venous congestion in Fontan patients can confound the use of liver stiffness as a biomarker for fibrosis as assessed by percentage of SR staining, Ishak score, Metavir score, and CHFS score. These results provide motivation for further development of magnetic resonance imaging-based biomarkers to increase the specificity in the assessment of Fontan-associated liver disease. A steady increase in liver stiffness observed in these patients may be useful for longitudinal follow-up of liver health., (Copyright © 2022 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.)

Background: Esophageal remodeling is a factor in disease progression and symptom severity for patients with eosinophilic esophagitis (EoE). Remodeling can begin early in children, resulting in stricture and food impaction. Detection of esophageal remodeling often depends on endoscopy and is appreciated only in its later stages., Objective: We sought to determine whether luminal eosinophil-associated and remodeling proteins captured by the esophageal string test (EST) correlate with measures of esophageal remodeling and biomarkers of the epithelial-mesenchymal transition (EMT)., Methods: Patients with EoE (7-18 years old) were enrolled from 2 pediatric hospitals. Participants performed the EST and underwent endoscopy. Histology, distensibility measured by endoluminal functional lumen imaging probe, and symptoms were assessed. Protein quantitation by ELISA was performed on mucosal biopsy and EST samples. Tissue sections were evaluated for EMT. Outcome measures were summarized, and Spearman ρ was used to assess bivariate correlations., Results: Forty patients (68% male) were enrolled (mean age, 12.5 years). Twenty-four (60%) had active disease (≥15 eosinophils per high-power field). EST-captured eotaxin-3, major basic protein 1, EDN, eosinophil peroxidase, and Charcot-Leyden crystal protein/galectin-10 showed significant correlations with peak eosinophils per high-power field (ρ 0.53-0.68, P < .001). Luminal proteins positively correlated with endoscopic features and markers of EMT, and negatively with esophageal distensibility. Periostin was captured by the EST and correlated with eosinophil density, basal zone hyperplasia, endoscopic appearance, and markers of EMT., Conclusion: Luminal markers of esophageal remodeling in addition to biomarkers of eosinophilic inflammation correlate with epithelial and functional remodeling in EoE., (Copyright © 2022 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Very early-onset inflammatory bowel disease (VEO-IBD), IBD diagnosed in children younger than 6 years old, is phenotypically and genetically distinct from older onset IBD. Monogenic and digenic causative defects, particularly in primary immunodeficiency and intestinal epithelial barrier genes, have been identified in a subset of patients with VEO-IBD allowing for targeted therapies and improved outcomes. However, these findings are the minority, thus strategies to correctly diagnose patients, including identification of specific histopathologic findings with correlating clinical and laboratory features may provide critical and necessary insight into mechanisms of disease pathogenesis and subsequent therapeutic options. In this article, we review the pathologic findings seen in patients with VEO-IBD and outline a pattern-based approach to diagnosis using examples from primary immunodeficiencies with gastrointestinal manifestations., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: L-kynurenine is a tryptophan-derived immunosuppressive metabolite and precursor to neurotoxic anthranilate and quinolinate. We evaluated the stereoisomer D-kynurenine as an immunosuppressive therapeutic which is hypothesized to produce less neurotoxic metabolites than L-kynurenine., Methods: L-/D-kynurenine effects on human and murine T cell function were examined in vitro and in vivo (homeostatic proliferation, colitis, cardiac transplant). Kynurenine effects on T cell metabolism were interrogated using [ 13 C] glucose, glutamine and palmitate tracing. Kynurenine was measured in tissues from human and murine tumours and kynurenine-fed mice., Findings: We observed that 1 mM D-kynurenine inhibits T cell proliferation through apoptosis similar to L-kynurenine. Mechanistically, [ 13 C]-tracing revealed that co-stimulated CD4 + T cells exposed to L-/D-kynurenine undergo increased β-oxidation depleting fatty acids. Replenishing oleate/palmitate restored effector T cell viability. We administered dietary D-kynurenine reaching tissue kynurenine concentrations of 19 μM, which is close to human kidney (6 μM) and head and neck cancer (14 μM) but well below the 1 mM required for apoptosis. D-kynurenine protected Rag1 -/- mice from autoimmune colitis in an aryl-hydrocarbon receptor dependent manner but did not attenuate more stringent immunological challenges such as antigen mismatched cardiac allograft rejection., Interpretation: Our dietary kynurenine model achieved tissue concentrations at or above human cancer kynurenine and exhibited only limited immunosuppression. Sub-suppressive kynurenine concentrations in human cancers may limit the responsiveness to indoleamine 2,3-dioxygenase inhibition evaluated in clinical trials., Funding: The study was supported by the NIH, the Else Kröner-Fresenius-Foundation, Laffey McHugh foundation, and American Society of Nephrology., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Multiple reports over the past 2 years have provided the first complete structural analyses for the essential yeast chromatin remodeler, RSC, providing elaborate molecular details for its engagement with the nucleosome. However, there still remain gaps in resolution, particularly within the many RSC subunits that harbor histone binding domains.Solving contacts at these interfaces is crucial because they are regulated by posttranslational modifications that control remodeler binding modes and function. Modifications are dynamic in nature often corresponding to transcriptional activation states and cell cycle stage, highlighting not only a need for enriched spatial resolution but also temporal understanding of remodeler engagement with the nucleosome. Our recent work sheds light on some of those gaps by exploring the binding interface between the RSC catalytic motor protein, Sth1, and the nucleosome, in the living nucleus. Using genetically encoded photo-activatable amino acids incorporated into histones of living yeast we are able to monitor the nucleosomal binding of RSC, emphasizing the regulatory roles of histone modifications in a spatiotemporal manner. We observe that RSC prefers to bind H2B SUMOylated nucleosomes in vivo and interacts with neighboring nucleosomes via H3K14ac. Additionally, we establish that RSC is constitutively bound to the nucleosome and is not ejected during mitotic chromatin compaction but alters its binding mode as it progresses through the cell cycle. Our data offer a renewed perspective on RSC mechanics under true physiological conditions.

A 11-year-old boy presented to the gastroenterology clinic after a 5-month history of fatigue, pallor, intermittent abdominal pain, and iron-deficiency anemia. Although the initial upper endoscopy was visually normally, the histological assessment was suggestive of eosinophilic gastritis. After multiple scopes and failed therapies, histologic analysis revealed a focus of thickened subepithelial collagen deposition suggestive of collagenous gastritis. A retrospective review of gastric biopsies using Gomori trichrome stain revealed previously unappreciated collagen deposition. This case report illustrates the benefit of performing trichrome stain on gastric biopsies in the setting of persistent or isolated gastric eosinophilia or iron deficiency anemia., (© 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

Alloimmune hemolytic disease of the fetus or newborn (HDFN) is a rare cause of neonatal cholestasis. HDFN-associated cholestasis has most often been reported secondary to anti-D alloimmunization. In utero transfusions are also an identified risk factor. A variety of diagnostic and therapeutic strategies have been described, mostly in case reports. Here, we report 2 cases of HDFN-associated cholestasis that were notable for extreme laboratory abnormalities including a peak ferritin of 24 700 ng/mL and a peak alanine aminotransferase of 1406 U/L (33.5-fold upper limit of normal). One case was due to alloimmunization other than anti-D. These cases help define the range of laboratory derangements that are consistent with HDFN-associated cholestasis, including extreme hyperferritinemia. Although in a number of cases, researchers have reported the use of iron chelation in these infants, herein, we describe successful management without iron chelation., Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2021 by the American Academy of Pediatrics.)

Chromatin remodeling complexes are multi-subunit nucleosome translocases that reorganize chromatin in the context of DNA replication, repair, and transcription. To understand how these complexes find their target sites on chromatin, we use genetically encoded photo-cross-linker amino acids to map the footprint of Sth1, the catalytic subunit of the RSC complex, on nucleosomes in living yeast. We find that H3 K14 acetylation induces the interaction of the Sth1 bromodomain with the H3 tail and mediates the interaction of RSC with neighboring nucleosomes rather than recruiting it to chromatin. RSC preferentially resides on H2B SUMOylated nucleosomes in vivo and shows a moderately enhanced affinity due to this modification in vitro. Furthermore, RSC is not ejected from chromatin in mitosis, but changes its mode of nucleosome binding. Our in vivo analyses show that RSC recruitment to specific chromatin targets involves multiple histone modifications likely in combination with histone variants and transcription factors., Competing Interests: Declaration of Interests The authors declare that no competing interests exist., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Small, 2-dimensional sections routinely used for human pathology analysis provide limited information about bowel innervation. We developed a technique to image human enteric nervous system (ENS) and other intramural cells in 3 dimensions., Methods: Using mouse and human colon tissues, we developed a method that combines tissue clearing, immunohistochemistry, confocal microscopy, and quantitative analysis of full-thickness bowel without sectioning to quantify ENS and other intramural cells in 3 dimensions., Results: We provided 280 adult human colon confocal Z-stacks from persons without known bowel motility disorders. Most of our images were of myenteric ganglia, captured using a 20× objective lens. Full-thickness colon images, viewed with a 10× objective lens, were as large as 4 × 5 mm 2 . Colon from 2 pediatric patients with Hirschsprung disease was used to show distal colon without enteric ganglia, as well as a transition zone and proximal pull-through resection margin where ENS was present. After testing a panel of antibodies with our method, we identified 16 antibodies that bind to molecules in neurons, glia, interstitial cells of Cajal, and muscularis macrophages. Quantitative analyses demonstrated myenteric plexus in 24.5% ± 2.4% of flattened colon Z-stack area. Myenteric ganglia occupied 34% ± 4% of myenteric plexus. Single myenteric ganglion volume averaged 3,527,678 ± 573,832 mm 3 with 38,706 ± 5763 neuron/mm 3 and 129,321 ± 25,356 glia/mm 3 . Images of large areas provided insight into why published values of ENS density vary up to 150-fold-ENS density varies greatly, across millimeters, so analyses of small numbers of thin sections from the same bowel region can produce varying results. Neuron subtype analysis revealed that approximately 56% of myenteric neurons stained with neuronal nitric oxide synthase antibody and approximately 33% of neurons produce and store acetylcholine. Transition zone regions from colon tissues of patients with Hirschsprung disease had ganglia in multiple layers and thick nerve fiber bundles without neurons. Submucosal neuron distribution varied among imaged colon regions., Conclusions: We developed a 3-dimensional imaging method for colon that provides more information about ENS structure than tissue sectioning. This approach could improve diagnosis for human bowel motility disorders and may be useful for other bowel diseases as well., (Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Gastrointestinal motility disorders include a spectrum of mild to severe clinical phenotypes that are caused by smooth muscle dysfunction. We investigated the genetic etiology of severe esophageal, gastric, and colonic dysmotility in two unrelated families with autosomal dominant disease presentation. Using exome sequencing, we identified a 2 base pair insertion at the end of the myosin heavy chain 11 (MYH11) gene in all affected members of Family 1 [NM&#95;001040113:c.5819&#95;5820insCA(p.Gln1941Asnfs*91)] and a 1 base pair deletion at the same genetic locus in Proband 2 [NM&#95;001040113:c.5819del(p.Pro1940Hisfs*91)]. Both variants are predicted to result in a similarly elongated protein product. Heterozygous dominant negative MYH11 pathogenic variants have been associated with thoracic aortic aneurysm and dissection while biallelic null alleles have been associated with megacystis microcolon intestinal hypoperistalsis syndrome. This report highlights heterozygous protein-elongating MYH11 variants affecting the SM2 isoforms of MYH11 as a cause for severe gastrointestinal dysmotility, and we hypothesize that the mechanistic pathogenesis of this disease, dominant hypercontractile loss-of-function, is distinct from those implicated in other diseases involving MYH11 dysfunction., (© 2020 Wiley Periodicals, Inc.)

Background & Aims: Eosinophilic esophagitis (EoE) is caused by an immune response to specific food allergens. There are no approved therapies beyond avoidance of the allergen(s) or treatment of inflammation. Epicutaneous immunotherapy (EPIT) reduces features of eosinophilic gastrointestinal disease in mice and pigs. We performed randomized, placebo-controlled study to determine the safety and efficacy of EPIT with Viaskin milk in children with milk-induced EoE., Methods: In a double-blind study, 20 children (4-17 years old) with milk-induced EoE were randomly assigned to groups given EPIT with Viaskin milk (n = 15) or placebo (n = 5) for 9 months during a milk-free period, followed by milk-containing diet for 2 months with EPIT. Then, subjects underwent upper endoscopy analysis, biopsies were collected, and maximum esophageal eosinophil counts were determined and was the primary endpoint. After upper endoscopy, patients were given open-label EPIT for 11 months (open-label phase). The subjects were allowed to consume milk if they had maximum values of fewer than 10 eosinophils/high-power field (eos/hpf); otherwise, they remained on a milk-free diet until the last 2 months of the open-label phase., Results: In the intent to treat population, there was no significant difference between the Viaskin milk group in mean eos/hpf (50.1 ± 43.97 eos/hpf) vs the placebo group (48.20 ± 56.98 eos/hpf). However, in the per-protocol population (7 patients given Viaskin milk and 2 patients given placebo), patients given Viaskin milk patients had a significantly lower mean eos/hpf count (25.57 ± 31.19) than patients given placebo (95.00 ± 63.64) (p = .038). At the end of the open-label phase, 9 of 19 evaluable subjects had mean values of fewer than 15 eos/hpf (47% response). The number of adverse events did not differ significantly between the Viaskin milk and placebo groups; there was 1 serious adverse event in the placebo group., Conclusions: In a pilot study of pediatric patients with EoE given EPIT with Viaskin milk or placebo for 11 months, we found no significant difference between groups for the maximum eosinophil count at the end of the study. However, findings from a per-protocol analysis indicate that Viaskin milk can reduce eos/hpf. At study completion, 47% of patients who continued open-label Viaskin milk for an additional 11 months had mean values of fewer than 15 eos/hpf. ClinicalTrials.gov no: NCT02579876., (Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Gastrointestinal polyps are mucosal overgrowths that, if unchecked, can undergo malignant transformation. Although relatively uncommon in the pediatric age group, they can be the harbingers of multiorgan cancer risk and require close management and follow-up. Additionally, as many polyposis syndromes are inherited, appropriate genetic testing and management of relatives is vital for the health of the entire family. In this review, we discuss both common and uncommon childhood gastrointestinal polyposis syndromes in terms of clinical presentation, management, and surveillance. We also detail any additional malignancy risk and surveillance required in the pediatric age group (<21 years old). Through this review, we provide a framework for gastroenterologists to manage the multifaceted nature of pediatric polyposis syndromes.

RNA binding proteins, including IMP1/IGF2BP1, are essential regulators of intestinal development and cancer. Imp1 hypomorphic mice exhibit gastrointestinal growth defects, yet the specific role for IMP1 in colon epithelial repair is unclear. Our prior work revealed that intestinal epithelial cell-specific Imp1 deletion ( Imp1 Δ IEC ) was associated with better regeneration in mice after irradiation. Here, we report increased IMP1 expression in patients with Crohn's disease and ulcerative colitis. We demonstrate that Imp1 Δ IEC mice exhibit enhanced recovery following dextran sodium sulfate (DSS)-mediated colonic injury. Imp1 Δ IEC mice exhibit Paneth cell granule changes, increased autophagy flux, and upregulation of Atg5. In silico and biochemical analyses revealed direct binding of IMP1 to MAP1LC3B , ATG3, and ATG5 transcripts. Genetic deletion of essential autophagy gene Atg7 in Imp1 Δ IEC mice revealed increased sensitivity of double-mutant mice to colonic injury compared to control or Atg7 single mutant mice, suggesting a compensatory relationship between Imp1 and the autophagy pathway. The present study defines a novel interplay between IMP1 and autophagy, where IMP1 may be transiently induced during damage to modulate colonic epithelial cell responses to damage., (© 2019 The Authors.)

Alterations in gut microbiota are known to affect intestinal inflammation and obesity. Antibiotic treatment can affect weight gain by elimination of histone deacetylase (HDAC) inhibitor-producing microbes, which are anti-inflammatory by augmenting regulatory T (T reg ) cells. We asked whether mice that lack HDAC6 and have potent suppressive T reg cells are protected from microbiota-induced accelerated weight gain. We crossed wild-type and HDAC6-deficient mice and subjected the offspring to perinatal penicillin, inducing weight gain via microbiota disturbance. We observed that male HDAC6-deficient mice were not protected and developed profoundly accelerated weight gain. The antibiotic-exposed HDAC6-deficient mice showed a mixed immune phenotype with increased CD4 + and CD8 + T-cell activation yet maintained the enhanced T reg cell-suppressive function phenotype characteristic of HDAC6-deficient mice. 16S rRNA sequencing of mouse fecal samples reveals that their microbiota diverged with time, with HDAC6 deletion altering microbiome composition. On a high-fat diet, HDAC6-deficient mice were depleted in representatives of the S24-7 family and Lactobacillus but enriched with Bacteroides and Parabacteroides; these changes are associated with obesity. Our findings further our understanding of the influence of HDACs on microbiome composition and are important for the development of HDAC6 inhibitors in the treatment of human diseases.-Lieber, A. D., Beier, U. H., Xiao, H., Wilkins, B. J., Jiao, J., Li, X. S., Schugar, R. C., Strauch, C. M., Wang, Z., Brown, J. M., Hazen, S. L., Bokulich, N. A., Ruggles, K. V., Akimova, T., Hancock, W. W., Blaser, M. J. Loss of HDAC6 alters gut microbiota and worsens obesity.

A fundamental challenge in understanding cardiac biology and disease is that the remarkable heterogeneity in cell type composition and functional states have not been well characterized at single-cell resolution in maturing and diseased mammalian hearts. Massively parallel single-nucleus RNA sequencing (snRNA-seq) has emerged as a powerful tool to address these questions by interrogating the transcriptome of tens of thousands of nuclei isolated from fresh or frozen tissues. snRNA-seq overcomes the technical challenge of isolating intact single cells from complex tissues, including the maturing mammalian hearts; reduces biased recovery of easily dissociated cell types; and minimizes aberrant gene expression during the whole-cell dissociation. Here we applied sNucDrop-seq, a droplet microfluidics-based massively parallel snRNA-seq method, to investigate the transcriptional landscape of postnatal maturing mouse hearts in both healthy and disease states. By profiling the transcriptome of nearly 20,000 nuclei, we identified major and rare cardiac cell types and revealed significant heterogeneity of cardiomyocytes, fibroblasts, and endothelial cells in postnatal developing hearts. When applied to a mouse model of pediatric mitochondrial cardiomyopathy, we uncovered profound cell type-specific modifications of the cardiac transcriptional landscape at single-nucleus resolution, including changes of subtype composition, maturation states, and functional remodeling of each cell type. Furthermore, we employed sNucDrop-seq to decipher the cardiac cell type-specific gene regulatory network (GRN) of GDF15, a heart-derived hormone and clinically important diagnostic biomarker of heart disease. Together, our results present a rich resource for studying cardiac biology and provide new insights into heart disease using an approach broadly applicable to many fields of biomedicine., (© 2018 Hu et al.; Published by Cold Spring Harbor Laboratory Press.)

Infants with congenital diarrheal disorders caused by enteroendocrine cell dysgenesis, or the loss of intestinal endocrine cells, causes severe malabsorptive diarrhea, though the mechanism is not fully understood. The transcription factor "aristaless-related homeobox" (Arx) is specifically expressed in intestinal endocrine cells. This study seeks to characterize the early malabsorptive phenotype of mice deficient for Arx using cell-type specific gene ablation in Villin-Cre; Arx loxP/Y ( Arx int ) mice. In neonatal mice, the loss of intestinal Arx caused the loss of intestinal hormones, such as cholecystokinin, secretin, neurotensin, glucose-dependent insulinotropic peptide, glucagon-like peptide (GLP)-1 and GLP-2 but also upregulation of somatostatin. Arx int mice exhibited steatorrhea with the loss of lipid transport in duodenal enterocytes, upregulation of lysozyme-positive Paneth cells, and a secondary increase in antimicrobial peptides, specifically Reg3β. When the epithelium from Arx int mice was cultured ex vivo into enteroids, however, the Reg3β upregulation was lost under the sterile conditions. Thus, Arx is required for the appropriate lineage allocation of multiple enteroendocrine subtypes. We concluded that altered hormonal signaling caused by Arx deficiency results in lipid malabsorption, premature Paneth cell differentiation, and an inflammatory response, including neutrophilic infiltrates and a microbiota-triggered upregulation of Reg3β. NEW & NOTEWORTHY The enteroendocrine transcription factor aristaless-related homeobox (Arx) plays a key role in lineage specification. Changes in hormonal expression mediated by Arx lead to lipid malabsorption and premature Paneth cell development. Furthermore, global profiling of whole intestine from Arx-deficient mice revealed significant upregulation of antimicrobial peptides. This antimicrobial response in Arx-deficient animals is lost under sterile culture conditions of enteroids.

Mitochondrial dysfunction is increasingly recognized as a critical determinant of both hereditary and acquired kidney diseases. However, it remains poorly understood how mitochondrial metabolism is regulated to support normal kidney function and how its dysregulation contributes to kidney disease. Here, we show that the nuclear receptor estrogen-related receptor gamma (ERRγ) and hepatocyte nuclear factor 1 beta (HNF1β) link renal mitochondrial and reabsorptive functions through coordinated epigenomic programs. ERRγ directly regulates mitochondrial metabolism but cooperatively controls renal reabsorption via convergent binding with HNF1β. Deletion of ERRγ in renal epithelial cells (RECs), in which it is highly and specifically expressed, results in severe renal energetic and reabsorptive dysfunction and progressive renal failure that recapitulates phenotypes of animals and patients with HNF1β loss-of-function gene mutations. Moreover, ERRγ expression positively correlates with renal function and is decreased in patients with chronic kidney disease (CKD). REC-ERRγ KO mice share highly overlapping renal transcriptional signatures with human patients with CKD. Together these findings reveal a role for ERRγ in directing independent and HNF1β-integrated programs for energy production and use essential for normal renal function and the prevention of kidney disease., Competing Interests: The authors declare no conflict of interest.

Objectives: Sequelae of eosinophilic esophagitis (EoE) include food impaction and esophageal stricture. Duration of inflammation is a predicted risk factor; however, complications remain unpredictable. Studies using the functional lumen imaging probe (FLIP) have demonstrated decreased distensibility of the esophagus in adult patients with EoE. As the impact of inflammation on the developing esophagus is unknown, we investigated esophageal distensibility in a pediatric cohort to determine the effect of age, ongoing inflammation, and fibrotic features on distensibility., Methods: We conducted a prospective observational study at two tertiary pediatric institutions. Subjects underwent FLIP evaluation during endoscopy to determine distensibility of the esophagus. During stepwise distension, simultaneous intrabag pressure and 16 channels of cross-sectional areas were measured. The minimal diameter at maximal esophageal distention at an intrabag pressure of 40 mm Hg was identified. Distensibility was compared between EoE and non-EoE subjects and between clinical variables within the EoE cohort. Potential confounding variables were identified., Results: Forty-four non-EoE and 88 EoE subjects aged 3-18 years were evaluated. Age positively correlated with esophageal distensibility in the non-EoE cohort, but this trend was not observed in the EoE population. Subjects with EoE had reduced distensibility even after adjusting for age. Active inflammation (eosinophils >15 eos/high-power field), histological lamina propria fibrosis, and various features of a fibrotic phenotype (stricture, food impaction, circumferential rings on endoscopy) were associated with decreased distensibility within the EoE cohort. FLIP was safe, feasible, and well tolerated., Conclusions: These findings suggest that remodeling occurs in the pediatric EoE population, warranting early diagnosis and initiation of therapy prior to the onset of disease complications.

The endocrine system is crucial for maintaining whole-body homeostasis. Little is known regarding endocrine hormones secreted by the heart other than atrial/brain natriuretic peptides discovered over 30 years ago. Here, we identify growth differentiation factor 15 (GDF15) as a heart-derived hormone that regulates body growth. We show that pediatric heart disease induces GDF15 synthesis and secretion by cardiomyocytes. Circulating GDF15 in turn acts on the liver to inhibit growth hormone (GH) signaling and body growth. We demonstrate that blocking cardiomyocyte production of GDF15 normalizes circulating GDF15 level and restores liver GH signaling, establishing GDF15 as a bona fide heart-derived hormone that regulates pediatric body growth. Importantly, plasma GDF15 is further increased in children with concomitant heart disease and failure to thrive (FTT). Together these studies reveal a new endocrine mechanism by which the heart coordinates cardiac function and body growth. Our results also provide a potential mechanism for the well-established clinical observation that children with heart diseases often develop FTT., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

Unlabelled: Biliatresone is an electrophilic isoflavone isolated from Dysphania species plants that has been causatively linked to naturally occurring outbreaks of a biliary atresia (BA)-like disease in livestock. Biliatresone has selective toxicity for extrahepatic cholangiocytes (EHCs) in zebrafish larvae. To better understand its mechanism of toxicity, we performed transcriptional profiling of liver cells isolated from zebrafish larvae at the earliest stage of biliatresone-mediated biliary injury, with subsequent comparison of biliary and hepatocyte gene expression profiles. Transcripts encoded by genes involved in redox stress response, particularly those involved in glutathione (GSH) metabolism, were among the most prominently up-regulated in both cholangiocytes and hepatocytes of biliatresone-treated larvae. Consistent with these findings, hepatic GSH was depleted at the onset of biliary injury, and in situ mapping of the hepatic GSH redox potential using a redox-sensitive green fluorescent protein biosensor showed that it was significantly more oxidized in EHCs both before and after treatment with biliatresone. Pharmacological and genetic manipulation of GSH redox homeostasis confirmed the importance of GSH in modulating biliatresone-induced injury given that GSH depletion sensitized both EHCs and the otherwise resistant intrahepatic cholangiocytes to the toxin, whereas replenishing GSH level by N-acetylcysteine administration or activation of nuclear factor erythroid 2-like 2 (Nrf2), a transcriptional regulator of GSH synthesis, inhibited EHC injury., Conclusion: These findings strongly support redox stress as a critical contributing factor in biliatresone-induced cholangiocyte injury, and suggest that variations in intrinsic stress responses underlie the susceptibility profile. Insufficient antioxidant capacity of EHCs may be critical to early pathogenesis of human BA. (Hepatology 2016;64:894-907)., (© 2016 by the American Association for the Study of Liver Diseases.)

Eosinophilic esophagitis (EoE) is a chronic Th2 and food antigen-mediated disease characterized by esophageal eosinophilic infiltration. Thymic stromal lymphopoetin (TSLP), an epithelial derived cytokine which bridges innate and Th2-type adaptive immune responses in other allergic conditions, is overexpressed in esophageal biopsies of EoE subjects. However, the triggers of TSLP expression in the esophageal epithelium are unknown. The objective of the current study was to characterize TSLP expression in human esophageal epithelium in EoE in vivo and to determine the role of food antigens upon epithelial TSLP expression in vitro. Using immunohistochemistry (IHC), we localized TSLP in esophageal biopsies of active EoE (≥15 eos/hpf), inactive EoE (<15 eos/hpf) and non-EoE control subjects, and found that TSLP expression was restricted to the differentiated suprabasal layer of the epithelium in actively inflamed EoE biopsies. Consistent with these results in vivo, inducible TSLP protein secretion was higher in CaCl2 differentiated telomerase-immortalized esophageal epithelial cells (EPC2-hTERT) compared to undifferentiated cells of the basal phenotype, following stimulation with the TLR3 ligand poly(I:C). To determine whether food antigens could directly induce epithelial TSLP secretion, differentiated and undifferentiated primary esophageal epithelial cells from EoE and non-EoE subjects were challenged with food antigens clinically relevant to EoE: Chicken egg ovalbumin (OVA), wheat, and milk proteins beta-lactoglobulin (blg) and beta-casein. Food antigens failed to induce TSLP secretion by undifferentiated cells; in contrast, only OVA induced TSLP secretion in differentiated epithelial cells from both EoE and control cell lines, an effect abolished by budesonide and NF-κb inhibition. Together, our study shows that specific food antigens can trigger innate immune mediated esophageal TSLP secretion, suggesting that esophageal epithelial cells at the barrier surface may play a significant role in the pathogenesis of EoE by regulating TSLP expression.

The segregation of eukaryotic chromosomes during mitosis requires their extensive folding into units of manageable size for the mitotic spindle. Here, we report on how phosphorylation at serine 10 of histone H3 (H3 S10) contributes to this process. Using a fluorescence-based assay to study local compaction of the chromatin fiber in living yeast cells, we show that chromosome condensation entails two temporally and mechanistically distinct processes. Initially, nucleosome-nucleosome interaction triggered by H3 S10 phosphorylation and deacetylation of histone H4 promote short-range compaction of chromatin during early anaphase. Independently, condensin mediates the axial contraction of chromosome arms, a process peaking later in anaphase. Whereas defects in chromatin compaction have no observable effect on axial contraction and condensin inactivation does not affect short-range chromatin compaction, inactivation of both pathways causes synergistic defects in chromosome segregation and cell viability. Furthermore, both pathways rely at least partially on the deacetylase Hst2, suggesting that this protein helps coordinating chromatin compaction and axial contraction to properly shape mitotic chromosomes.

Increasing evidence indicates that the gut microbiota can be altered to ameliorate or prevent disease states, and engineering the gut microbiota to therapeutically modulate host metabolism is an emerging goal of microbiome research. In the intestine, bacterial urease converts host-derived urea to ammonia and carbon dioxide, contributing to hyperammonemia-associated neurotoxicity and encephalopathy in patients with liver disease. Here, we engineered murine gut microbiota to reduce urease activity. Animals were depleted of their preexisting gut microbiota and then inoculated with altered Schaedler flora (ASF), a defined consortium of 8 bacteria with minimal urease gene content. This protocol resulted in establishment of a persistent new community that promoted a long-term reduction in fecal urease activity and ammonia production. Moreover, in a murine model of hepatic injury, ASF transplantation was associated with decreased morbidity and mortality. These results provide proof of concept that inoculation of a prepared host with a defined gut microbiota can lead to durable metabolic changes with therapeutic utility.

Heritable and acquired biliary disorders are an important cause of acute and chronic human liver disease. Biliary development and physiology have been studied extensively in rodent models and more recently, zebrafish have been used to uncover pathogenic mechanisms and potential therapies for these conditions. Here we report development of novel transgenic lines labeling the intrahepatic and extrahepatic biliary system of zebrafish larvae that can be used for lineage tracing and isolation of biliary-specific RNAs from mixed populations of liver cells. We show that GFP expression driven by a 4.4 kilobase promoter fragment from the zebrafish keratin18 (krt18) gene allows visualization of all components of the developing biliary system as early as 3 days post-fertilization. In addition, expression of a ribosomal fusion protein (EGFP-Rpl10a) in krt18:TRAP transgenic fish allows for enrichment of translated biliary cell mRNAs via translating ribosome affinity purification (TRAP). Future studies utilizing these reagents will enhance our understanding of the morphologic and molecular processes involved in biliary development and disease., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Metaphase chromosomes are visible hallmarks of mitosis, yet our understanding of their structure and of the forces shaping them is rudimentary. Phosphorylation of histone H3 serine 10 (H3 S10) by Aurora B kinase is a signature event of mitosis, but its function in chromatin condensation is unclear. Using genetically encoded ultraviolet light-inducible cross-linkers, we monitored protein-protein interactions with spatiotemporal resolution in living yeast to identify the molecular details of the pathway downstream of H3 S10 phosphorylation. This modification leads to the recruitment of the histone deacetylase Hst2p that subsequently removes an acetyl group from histone H4 lysine 16, freeing the H4 tail to interact with the surface of neighboring nucleosomes and promoting fiber condensation. This cascade of events provides a condensin-independent driving force of chromatin hypercondensation during mitosis.

North American Indian Childhood Cirrhosis (NAIC) is a rare, autosomal recessive, progressive cholestatic disease of infancy affecting the Cree-Ojibway first Nations of Quebec. All NAIC patients are homozygous for a missense mutation (R565W) in CIRH1A, the human homolog of the yeast nucleolar protein Utp4. Utp4 is part of the t-Utp subcomplex of the small subunit (SSU) processome, a ribonucleoprotein complex required for ribosomal RNA processing and small subunit assembly. NAIC has thus been proposed to be a primary ribosomal disorder (ribosomopathy); however, investigation of the pathophysiologic mechanism of this disease has been hindered by lack of an animal model. Here, using a morpholino oligonucleotide (MO)-based loss-of-function strategy, we have generated a model of NAIC in the zebrafish, Danio rerio. Zebrafish Cirhin shows substantial homology to the human homolog, and cirh1a mRNA is expressed in developing hepatocytes and biliary epithelial cells. Injection of two independent MOs directed against cirh1a at the one-cell stage causes defects in canalicular and biliary morphology in 5 dpf larvae. In addition, 5 dpf Cirhin-deficient larvae have dose-dependent defects in hepatobiliary function, as assayed by the metabolism of an ingested fluorescent lipid reporter. Previous yeast and in vitro studies have shown that defects in ribosome biogenesis cause stabilization and nuclear accumulation of p53, which in turn causes p53-mediated cell cycle arrest and/or apoptosis. Thus, the nucleolus appears to function as a cellular stress sensor in some cell types. In accordance with this hypothesis, transcriptional targets of p53 are upregulated in Cirhin-deficient zebrafish embryos, and defects in biliary function seen in Cirhin-deficient larvae are completely abrogated by mutation of tp53. Our data provide the first in vivo evidence of a role for Cirhin in biliary development, and support the hypothesis that congenital defects affecting ribosome biogenesis can activate a cellular stress response mediated by p53.

The liver performs a large number of essential synthetic and regulatory functions that are acquired during fetal development and persist throughout life. Their disruption underlies a diverse group of heritable and acquired diseases that affect both pediatric and adult patients. Although experimental analyses used to study liver development and disease are typically performed in cell culture models or rodents, the zebrafish is increasingly used to complement discoveries made in these systems. Forward and reverse genetic analyses over the past two decades have shown that the molecular program for liver development is largely conserved between zebrafish and mammals, and that the zebrafish can be used to model heritable human liver disorders. Recent work has demonstrated that zebrafish can also be used to study the mechanistic basis of acquired liver diseases. Here, we provide a comprehensive summary of how the zebrafish has contributed to our understanding of human liver development and disease., (© 2013 American Physiological Society.)

Current biosynthetic methods for producing proteins containing site-specifically incorporated unnatural amino acids are inefficient because the majority of the amino acid goes unused. Here we present a universal approach to improve the efficiency of such processes using condensed Escherichia coli cultures., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)