ZIP8 is a metal transporter with a role in manganese (Mn) homeostasis. A common genetic variant in ZIP8 (rs13107325; A391T) ranks in the top 10 of pleiotropic single nucleotide polymorphisms (SNP) identified in genome-wide association studies, including associations with an increased risk of schizophrenia, obesity, Crohn’s disease, and reduced blood Mn. Here, we used CRISPR/Cas9-mediated knock-in (KI) to generate a mouse model of ZIP8 A391T (mouse Zip8 393T-KI). Recapitulating the SNP association with blood Mn, blood Mn is reduced in Zip8 393T-KI mice. There is restricted abnormal tissue Mn homeostasis with decreases in liver and kidney Mn and reciprocal increase in biliary Mn to provide in vivo evidence of hypomorphic Zip8 function. Upon challenge in a chemical-induced colitis model, male Zip8 393T-KI mice exhibited enhanced disease susceptibility. ZIP8 391-Thr associated with reduced triantennary plasma N-glycan species in a population-based cohort to define a genotype-specific glycophenotype hypothesized to be linked to Mn-dependent glycosyltransferase activity. This glycophenotype was maintained in a cohort of Crohn’s disease patients. These data and the pleiotropic disease associations with ZIP8 391-Thr suggest underappreciated roles of Mn homeostasis in compex human disease.
Laxmi Sunuwar, Azra Frkatovic, Sodbo Sharapov, Qinchuan Wang, Heather Neu, Xinqun Wu, Talin Haritunians, Fengyi Wan, Sarah L. J. Michel, Shaoguang Wu, Dermot McGovern, Gordan Lauc, Mark Donowitz, Cynthia L. Sears, Joanna M.P. Melia
Lynch syndrome is the most common colorectal cancer (CRC) hereditary form and it is characterized by DNA mismatch repair (MMR) deficiency. The term Lynch-like syndrome (LLS) is used for patients with MMR-deficient tumors and neither germline mutation in MLH1, MSH2, MSH6, PMS2, or EPCAM, nor MLH1 somatic methylation. Biallelic somatic inactivation or cryptic germline MMR variants undetected during genetic testing have been proposed to be involved. Sixteen patients with early-onset LLS CRC were selected for germline and tumor whole-exome sequencing. Two potentially pathogenic germline MCM8 variants were detected in a LLS male patient with fertility problems. A knockout cellular model for MCM8 was generated by CRISPR-Cas9 and detected genetic variants were produced by mutagenesis. DNA damage, microsatellite instability and mutational signatures were monitored. DNA damage was evident for MCM8KO cells and the analyzed genetic variants. Microsatellite instability and mutational signatures in MCM8KO cells were compatible with the involvement of MCM8 in MMR. Replication in an independent familial cancer cohort detected additional carriers. Unexplained MMR-deficient CRC cases, even showing somatic biallelic MMR inactivation, may be caused by underlying germline defects in genes different than the MMR genes. We suggest MCM8 as a new gene involved in CRC germline predisposition with a recessive pattern of inheritance.
Mariano Golubicki, Laia Bonjoch, José G. Acuña-Ochoa, Marcos Díaz-Gay, Jenifer Muñoz, Miriam Cuatrecasas, Teresa Ocaña, Soledad Iseas, Guillermo Mendez, Daniel Cisterna, Stephanie A. Schubert, Maartje Nielsen, Tom van Wezel, Yael Goldberg, Eli Pikarsky, Juan Robbio, Enrique Roca, Antoni Castells, Francesc Balaguer, Marina Antelo, Sergi Castellví-Bel
Actin γ 2, smooth muscle (ACTG2) R257C mutation is the most common genetic cause of visceral myopathy. Individuals with ACTG2 mutations endure prolonged hospitalizations and surgical interventions, become dependent on intravenous nutrition and bladder catheterization, and often die in childhood. Currently, we understand little about how ACTG2 mutations cause disease, and there are no mechanism-based treatments. Our goal was to characterize the effects of ACTG2R257C on actin organization and function in visceral smooth muscle cells. We overexpressed ACTG2WT or ACTG2R257C in primary human intestinal smooth muscle cells (HISMCs) and performed detailed quantitative analyses to examine effects of ACTG2R257C on (a) actin filament formation and subcellular localization, (b) actin-dependent HISMC functions, and (c) smooth muscle contractile gene expression. ACTG2R257C resulted in 41% fewer, 13% thinner, 33% shorter, and 40% less branched ACTG2 filament bundles compared with ACTG2WT. Curiously, total F-actin probed by phalloidin and a pan-actin antibody was unchanged between ACTG2WT- and ACTG2R257C-expressing HISMCs, as was ultrastructural F-actin organization. ACTG2R257C-expressing HISMCs contracted collagen gels similar to ACTG2WT-expressing HISMCs but spread 21% more and were 11% more migratory. In conclusion, ACTG2R257C profoundly affects ACTG2 filament bundle structure, without altering global actin cytoskeleton in HISMCs.
Sohaib Khalid Hashmi, Vasia Barka, Changsong Yang, Sabine Schneider, Tatyana M. Svitkina, Robert O. Heuckeroth
Shwachman-Diamond syndrome (SDS) is characterized by exocrine pancreatic insufficiency, neutropenia, and skeletal abnormalities. Biallelic mutations in SBDS, which encodes a ribosome maturation factor, are found in 90% of SDS cases. Sbds-/- mice are embryonic lethal. Using CRISPR/Cas9 editing, we created sbds-deficient zebrafish strains. Sbds protein levels progressively decreased and became undetectable at 10 days post fertilization (dpf). Polysome analysis revealed decreased 80S ribosomes. Homozygous mutant fish developed normally until 15 dpf. Mutant fish subsequently have stunted growth and shows signs of atrophy in pancreas, liver, and intestine. In addition, neutropenia occurred by 5 dpf. Upregulation of tp53 mRNA did not occur until 10 dpf and inhibition of proliferation correlating with death by 21 dpf. Transcriptome analysis showed tp53 activation through upregulation of genes involved in cell cycle arrest, cdkn1a and ccng1, and apoptosis, puma and mdm2. However, elimination of Tp53 function did not prevent lethality. Because of growth retardation and atrophy of intestinal epithelia, we studied the effects of starvation on wildtype fish. Starved wildtype fish showed intestinal atrophy, zymogen granule loss, and tp53 upregulation – similar to the mutant phenotype. In addition, there was reduction in neutral lipid storage and ribosomal protein amount, similar to the mutant phenotype. Thus, loss of Sbds in zebrafish phenocopies much of the human disease and is associated with growth arrest and tissue atrophy, particularly of the gastrointestinal system, at the larval stage. A variety of stress responses, some associated with Tp53, contribute to pathophysiology of SDS.
Usua Oyarbide, Arish N. Shah, Wilmer Amaya-Mejia, Matthew Snyderman, Margaret Kell, Daniela Allende, Eliezer Calo, Jacek Topczewski, Seth Corey
Allergic disorders, characterized by Th2 immune responses to environmental substances, are increasingly common in children in Western societies. Multiple studies indicate that breastfeeding, early complementary introduction of food allergens, and antibiotic avoidance in the first year of life reduces allergic outcomes in at-risk children. Why the benefit of these practices is restricted to early life is largely unknown. We identified a preweaning interval during which dietary antigens are assimilated by the colonic immune system. This interval is under maternal control via temporal changes in breast milk, coincides with an influx of naive T cells into the colon, and is followed by the development of a long-lived population of colonic peripherally derived Tregs (pTregs) that can be specific for dietary antigens encountered during this interval. Desynchronization of mothers and offspring produced durable deficits in these pTregs, impaired tolerance to dietary antigens introduced during and after this preweaning interval, and resulted in spontaneous Th2 responses. These effects could be rescued by pTregs from the periweaning colon or by Tregs generated in vitro using periweaning colonic antigen-presenting cells. These findings demonstrate that mothers and their offspring are synchronized for the development of a balanced immune system.
Kathryn A. Knoop, Keely G. McDonald, Paige E. Coughlin, Devesha H. Kulkarni, Jenny K. Gustafsson, Brigida Rusconi, Vini John, I. Malick Ndao, Avraham Beigelman, Misty Good, Barbara B. Warner, Charles O. Elson, Chyi-Song Hsieh, Simon P. Hogan, Phillip I. Tarr, Rodney D. Newberry
The aim of this study was to elucidate the role and the pathways used by bile acid receptor TGR5 in transmitting satiety signals. We showed TGR5 colocalized with cholecystokinin type A (CCK-A) receptors in a subpopulation of rat nodose ganglia (NG) neurons. Intra-arterial injection of deoxycholic acid (DCA) dose-dependently increased firing rate in NG while a subthreshold dose of DCA and CCK-8 increased firing rates synergistically. TGR5-specific agonist oleanolic acid induced NG neuronal firing in a dose-dependent manner. However, the same units did not respond to GW4064, a nuclear receptor–specific agonist. Quantity of DCA-activated neurons in the hypothalamus was determined by c-Fos expression. Combining DCA and CCK-8 caused a 4-fold increase in c-Fos activation. In the arcuate nucleus, c-Fos–positive neurons coexpressed cocaine and amphetamine regulated transcript and proopiomelanocortin. DCA-induced c-Fos expression was eliminated following truncal vagotomy or silencing of TGR5 in the NG. Feeding studies showed intravenous injection of 1 μg/kg of DCA reduced food intake by 12% ± 3%, 24% ± 5%, and 32% ± 6% in the first 3 hours, respectively. Silencing of TGR5 or CCK-A receptor in the NG enhanced spontaneous feeding by 18% ± 2% and 13.5% ± 2.4%, respectively. When both TGR5 and CCK-A receptor were silenced, spontaneous feeding was enhanced by 37% ± 4% in the first 3 hours, suggesting that bile acid may have a physiological role in regulating satiety. Working in concert with CCK, bile acid synergistically enhanced satiety signals to reduce spontaneous feeding.
Xiaoyin Wu, Ji-Yao Li, Allen Lee, Yuan-Xu Lu, Shi-Yi Zhou, Chung Owyang
The regulatory mechanisms enabling the intestinal epithelium to maintain a high degree of regenerative capacity during mucosal injury remain unclear. Ex vivo survival and clonogenicity of intestinal stem cells (ISCs) strictly required Cdc42-mediated growth response and Cdc42-deficient enteroids undergo rapid apoptosis. Mechanistically, Cdc42 engaging with EGFR was required for EGF-stimulated receptor-mediated endocytosis and sufficient to promote MAPK signaling. Proteomics and kinase analysis revealed that a physiological, but non-conventionally, spliced Cdc42 variant 2 (V2), exhibited stronger MAPK-activating capability. Human CDC42-V2 is transcriptionally elevated in some colon tumor tissues. Accordingly, mice engineered to overexpress Cdc42-V2 in intestinal epithelium showed elevated MAPK signaling, enhanced regeneration, and reduced mucosal damage in response to irradiation. Overproducing Cdc42-V2 specifically in mouse ISCs enhanced intestinal regeneration following injury. Thus, the intrinsic Cdc42-MAPK program is required for intestinal epithelial regeneration while elevating this signaling cascade is capable of initiating protection from genotoxic injury.
Xiao Zhang, Sheila Bandyopadhyay, Leandro P. Araujo, Kevin Tong, Juan Flores, Daniel Laubitz, Yanlin Zhao, George Yap, Jingren Wang, Qingze Zou, Ronaldo P. Ferraris, Lanjing Zhang, Wenwei Hu, Edward M. Bonder, Pawel R. Kiela, Robert J. Coffey, Michael Verzi, Ivaylo I. Ivanov, Nan Gao
Arrestin domain containing 3 (ARRDC3) represents a newly discovered α-arrestin involved in obesity, inflammation and cancer. Here we demonstrated a pro-inflammation role of ARRDC3 in H. pylori-associated gastritis. Increased ARRDC3 was detected in gastric mucosa of patients and mice infected with H. pylori. ARRDC3 in gastric epithelial cells (GECs) was induced by H. pylori, regulated by ERK and PI3K-AKT pathways in a cagA-dependent manner. Human gastric ARRDC3 correlated with the severity of gastritis, and mouse ARRDC3 from non-BM-derived cells promoted gastric inflammation. This inflammation was characterized by the CXCR2-dependent influx of CD45+CD11b+Ly6C-Ly6G+ neutrophils, whose migration was induced via the ARRDC3-dependent production of CXCL2 by GECs. Importantly, gastric inflammation was attenuated in ARRDC3-/- mice but increased in protease-activated receptor 1 (PAR1)-/- mice. Mechanistically, ARRDC3 in GECs directly interacted with PAR1 and negatively regulated PAR1 via ARRDC3-mediated lysosomal degradation, which abrogated the suppression of CXCL2 production and following neutrophil chemotaxis by PAR1, thereby contributing to the development of H. pylori-associated gastritis. This study identifies a novel regulatory network involving H. pylori, GECs, ARRDC3, PAR1, and neutrophils, which collectively exert a pro-inflammatory effect within gastric microenvironment. Efforts to inhibit this ARRDC3-dependent pathway may prove valuable strategies in treating of H. pylori-associated gastritis.
Yu-gang Liu, Yong-sheng Teng, Zhi-guo Shan, Ping Cheng, Chuan-jie Hao, Yi-pin Lv, Fang-yuan Mao, Shi-ming Yang, Weisan Chen, Yong-Liang Zhao, Nan You, Quan-ming Zou, Yuan Zhuang
One of the major challenges in using pancreatic cancer patient-derived organoids (PDOs) in precision oncology is the time from biopsy to functional characterization. This is particularly true for biopsy specimen with limited tumor cell yield, typical characteristics of biopsies from endoscopic ultrasound-guided fine needle aspirations (EUS-FNAs).Here, we tested conditioned media of individual PDOs for cell-free tumor DNA (cfDNA) to detect driver mutations already early on during the expansion process in order to accelerate the genetic characterization of PDOs as well as subsequent functional testing. Importantly, genetic alterations detected in the PDO supernatant, collected as early as 72h after biopsy, recapitulate the mutational profile of the primary tumor indicating suitability of this approach to subject PDOs to drug testing in a reduced timeframe. In addition, we demonstrate that this workflow is practicable even in patients of whom the amount of tumor material was not sufficient for molecular characterization by established means.Our findings demonstrate that generating PDOs from very limited biopsy material permits molecular profiling and drug testing. With our approach this can be achieved in a rapid and feasible fashion with broad implications in clinical practice.
Zahra Dantes, Hsi-Yu Yen, Nicole Pfarr, Christof Winter, Katja Steiger, Alexander Muckenhuber, Alexander Hennig, Sebastian Lange, Thomas Engleitner, Rupert Öllinger, Roman Maresch, Felix Orben, Irina Heid, Georgios A. Kaissis, Kuangyu Shi, Geoffrey J. Topping, Fabian Stögbauer, Matthias Wirth, Katja Peschke, Aristeidis Papargyriou, Massoud Rezaee-Oghazi, Karin Feldmann, Arlett P. G. Schäfer, Raphela Ranjan, Clara Lubeseder-Martellato, Daniel E. Stange, Thilo Welsch, Marc E. Martignoni, Güralp Onur Ceyhan, Helmut Friess, Alexander Herner, Lucia Liotta, Matthias Treiber, Guido von Figura, Mohamed Abdelhafez, Peter Klare, Christoph Schlag, Hana Algül, Jens T. Siveke, Rickmer F. Braren, Gregor Weirich, Wilko Weichert, Dieter Saur, Roland Rad, Roland Schmid, Günter Schneider, Maximilian Reichert
Inflammation is a major risk factor of morbidity and mortality in older adults. Although its precise etiology is unknown, low-grade inflammation in older adults is commonly associated with increased intestinal epithelial permeability (leaky gut) and abnormal (dysbiotic) gut microbiota. The increasing older population and lack of treatments to reduce aging-related microbiota dysbiosis, leaky gut and inflammation culminates on a rise in aging-related comorbidities, constituting a significant public health concern. Here we demonstrate that a human-origin probiotic cocktail containing 5-Lactobacillus and 5 Enterococcus strains isolated from healthy infant’s gut prevents high-fat diet (HFD)-induced microbiota dysbiosis, leaky gut, inflammation, metabolic dysfunctions and physical function decline in older mice. Probiotic-modulated gut microbiota primarily reduced leaky gut by increasing tight junctions, which in turn reduced inflammation. Mechanistically, probiotics modulated microbiota in a way to increases bile salt hydrolase activity, which in turn increased taurine abundance in the gut that stimulated tight junctions and suppressed gut leakiness. Further, in Caenorhabditis elegans, taurine increased life span, reduced adiposity and leaky gut, and enhanced physical function. The results suggest that such probiotic therapies could prevent or treat aging-related leaky gut and inflammation in elderly.
Shokouh Ahmadi, Shaohua Wang, Ravinder Nagpal, Bo Wang, Shalini Jain, Atefeh Razazan, Sidharth P. Mishra, Xuewei Zhu, Zhan Wang, Kylie Kavanagh, Hariom Yadav
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