Cory J Smith, Oscar Castanon, Khaled Said, Verena Volf, Parastoo Khoshakhlagh, Amanda Hornick, Raphael Ferreira, Chun-Ting Wu, Marc Güell, Shilpa Garg, Alex HM Ng, Hannu Myllykallio, and George M Church. 2020. “Enabling large-scale genome editing at repetitive elements by reducing DNA nicking.” Nucleic Acids Res, 48, 9, Pp. 5183-5195.Abstract
To extend the frontier of genome editing and enable editing of repetitive elements of mammalian genomes, we made use of a set of dead-Cas9 base editor (dBE) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks and single-strand breaks. We used a set of gRNAs targeting repetitive elements-ranging in target copy number from about 32 to 161 000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ∼13 200 and ∼12 200 loci in 293T and human induced pluripotent stem cells (hiPSCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering.
Zhihui Wang, Mulong Du, Qianyu Yuan, Yichen Guo, John N Hutchinson, Li Su, Yinan Zheng, Jun Wang, Lorelei A Mucci, Xihong Lin, Lifang Hou, and David C Christiani. 2020. “Epigenomic analysis of 5-hydroxymethylcytosine (5hmC) reveals novel DNA methylation markers for lung cancers.” Neoplasia, 22, 3, Pp. 154-161.Abstract
BACKGROUND: DNA methylation at the fifth position of cytosine (5mC) is a common epigenetic alteration affecting a range of cellular processes. In recent years, 5-hydroxymethylcytosine (5hmC), an oxidized form of 5mC, has risen broad interests as a potential biomarker for lung cancer diagnosis and survival. METHODS: We analyzed the epigenome-wide 5hmC profiles of paired lung tumor and adjacent normal tissues, using the TET-Assisted Bisulfite (TAB) array - Infinium MethylationEPIC BeadChip (EPIC) approach. The differentially methylated CpG sites were identified, and the biological relevance of 5hmC was assessed by differential methylation regions (DMR) analysis and gene set analysis (GSA). RESULTS: We observed global hypomethylation of 5hmC comparing tumor to normal tissues, and hypermethylated 5hmC were enriched in CpG islands and gene upstream. Comparison of 5hmC and 5modC (total methylation: 5mC + 5hmC) profiling showed low correlation, and only a small proportion of the significant 5hmC loci overlapped with the significant total methylation loci. GSA analysis suggested that 5hmC was mainly involved in biological processes such as cellular process, biological regulation, and metabolic process. CONCLUSION: This is the first study to analyze the epigenome-wide 5hmC profiles among paired lung tumor and normal tissues. We observed global hypomethylation of 5hmC in lung cancers, and hypermethylated 5hmC enriched in CpG islands and gene upstream. We found that the genome-wide 5hmC levels do not correlate with the total methylation, and the GSA suggested different biological functions of 5hmC compared to 5modC. Overall, our results demonstrate the potential of 5hmC as a novel biomarker for lung cancer.
Misty R Riddle, Ariel C Aspiras, Fleur Damen, John N Hutchinson, Daniel J-F Chinnapen, Julius Tabin, and Clifford J Tabin. 2020. “Genetic architecture underlying changes in carotenoid accumulation during the evolution of the blind Mexican cavefish, Astyanax mexicanus.” J Exp Zool B Mol Dev Evol, 334, 7-8, Pp. 405-422.Abstract
Carotenoids are lipid-soluble yellow to orange pigments produced by plants, bacteria, and fungi. They are consumed by animals and metabolized to produce molecules essential for gene regulation, vision, and pigmentation. Cave animals represent an interesting opportunity to understand how carotenoid utilization evolves. Caves are devoid of light, eliminating primary production of energy through photosynthesis and, therefore, limiting carotenoid availability. Moreover, the selective pressures that favor carotenoid-based traits, like pigmentation and vision, are relaxed. Astyanax mexicanus is a species of fish with multiple river-adapted (surface) and cave-adapted populations (i.e., Tinaja, Pachón, Molino). Cavefish exhibit regressive features, such as loss of eyes and melanin pigment, and constructive traits, like increased sensory neuromasts and starvation resistance. Here, we show that, unlike surface fish, Tinaja and Pachón cavefish accumulate carotenoids in the visceral adipose tissue. Carotenoid accumulation is not observed in Molino cavefish, indicating that it is not an obligatory consequence of eye loss. We used quantitative trait loci mapping and RNA sequencing to investigate genetic changes associated with carotenoid accumulation. Our findings suggest that multiple stages of carotenoid processing may be altered in cavefish, including absorption and transport of lipids, cleavage of carotenoids into unpigmented molecules, and differential development of intestinal cell types involved in carotenoid assimilation. Our study establishes A. mexicanus as a model to study the genetic basis of natural variation in carotenoid accumulation and how it impacts physiology.
Dmitry Prokopenko, Julian Hecker, Rory Kirchner, Brad A Chapman, Oliver Hoffman, Kristina Mullin, Winston Hide, Lars Bertram, Nan Laird, Dawn L DeMeo, Christoph Lange, and Rudolph E Tanzi. 2020. “Identification of Novel Alzheimer's Disease Loci Using Sex-Specific Family-Based Association Analysis of Whole-Genome Sequence Data.” Sci Rep, 10, 1, Pp. 5029.Abstract
With the advent of whole genome-sequencing (WGS) studies, family-based designs enable sex-specific analysis approaches that can be applied to only affected individuals; tests using family-based designs are attractive because they are completely robust against the effects of population substructure. These advantages make family-based association tests (FBATs) that use siblings as well as parents especially suited for the analysis of late-onset diseases such as Alzheimer's Disease (AD). However, the application of FBATs to assess sex-specific effects can require additional filtering steps, as sensitivity to sequencing errors is amplified in this type of analysis. Here, we illustrate the implementation of robust analysis approaches and additional filtering steps that can minimize the chances of false positive-findings due to sex-specific sequencing errors. We apply this approach to two family-based AD datasets and identify four novel loci (GRID1, RIOK3, MCPH1, ZBTB7C) showing sex-specific association with AD risk. Following stringent quality control filtering, the strongest candidate is ZBTB7C (P = 1.83 × 10), in which the minor allele of rs1944572 confers increased risk for AD in females and protection in males. ZBTB7C encodes the Zinc Finger and BTB Domain Containing 7C, a transcriptional repressor of membrane metalloproteases (MMP). Members of this MMP family were implicated in AD neuropathology.
Georgios Theocharidis, Dimitrios Baltzis, Matthieu Roustit, Ana Tellechea, Seema Dangwal, Radhika S Khetani, Bin Shu, Wanni Zhao, Jianfang Fu, Swati Bhasin, Antonios Kafanas, Daniel Hui, Shannan Ho Sui, Nikolaos A Patsopoulos, Manoj Bhasin, and Aristidis Veves. 2020. “Integrated Skin Transcriptomics and Serum Multiplex Assays Reveal Novel Mechanisms of Wound Healing in Diabetic Foot Ulcers.” Diabetes, 69, 10, Pp. 2157-2169.Abstract
Nonhealing diabetic foot ulcers (DFUs) are characterized by low-grade chronic inflammation, both locally and systemically. We prospectively followed a group of patients who either healed or developed nonhealing chronic DFUs. Serum and forearm skin analysis, both at the protein expression and the transcriptomic level, indicated that increased expression of factors such as interferon-γ (IFN-γ), vascular endothelial growth factor, and soluble vascular cell adhesion molecule-1 were associated with DFU healing. Furthermore, foot skin single-cell RNA sequencing analysis showed multiple fibroblast cell clusters and increased inflammation in the dorsal skin of patients with diabetes mellitus (DM) and DFU specimens compared with control subjects. In addition, in myeloid cell DM and DFU upstream regulator analysis, we observed inhibition of interleukin-13 and IFN-γ and dysregulation of biological processes that included cell movement of monocytes, migration of dendritic cells, and chemotaxis of antigen-presenting cells pointing to an impaired migratory profile of immune cells in DM skin. The and genes, which were upregulated at the forearm of nonhealers, were mainly expressed by the vascular endothelial cell cluster almost exclusively in DFU, indicating a potential important role in wound healing. These results from integrated protein and transcriptome analyses identified individual genes and pathways that can potentially be targeted for enhancing DFU healing.
Pritha Sen, Adrian R Wilkie, Fei Ji, Yiming Yang, Ian J Taylor, Miguel Velazquez-Palafox, Emilia AH Vanni, Jean M Pesola, Rosio Fernandez, Han Chen, Liza M Morsett, Erik R Abels, Mary Piper, Rebekah J Lane, Suzanne E Hickman, Terry K Means, Eric S Rosenberg, Ruslan I Sadreyev, Bo Li, Donald M Coen, Jay A Fishman, and Joseph El Khoury. 2020. “Linking indirect effects of cytomegalovirus in transplantation to modulation of monocyte innate immune function.” Sci Adv, 6, 17, Pp. eaax9856.Abstract
Cytomegalovirus (CMV) is an important cause of morbidity and mortality in the immunocompromised host. In transplant recipients, a variety of clinically important "indirect effects" are attributed to immune modulation by CMV, including increased mortality from fungal disease, allograft dysfunction and rejection in solid organ transplantation, and graft-versus-host-disease in stem cell transplantation. Monocytes, key cellular targets of CMV, are permissive to primary, latent and reactivated CMV infection. Here, pairing unbiased bulk and single cell transcriptomics with functional analyses we demonstrate that human monocytes infected with CMV do not effectively phagocytose fungal pathogens, a functional deficit which occurs with decreased expression of fungal recognition receptors. Simultaneously, CMV-infected monocytes upregulate antiviral, pro-inflammatory chemokine, and inflammasome responses associated with allograft rejection and graft-versus-host disease. Our study demonstrates that CMV modulates both immunosuppressive and immunostimulatory monocyte phenotypes, explaining in part, its paradoxical "indirect effects" in transplantation. These data could provide innate immune targets for the stratification and treatment of CMV disease.
Stefan Haemmig, Dafeng Yang, Xinghui Sun, Debapria Das, Siavash Ghaffari, Roberto Molinaro, Lei Chen, Yihuan Deng, Dan Freeman, Norman Moullan, Yevgenia Tesmenitsky, Khyrul AKM Wara, Viorel Simion, Eugenia Shvartz, James F Lee, Tianlun Yang, Galina Sukova, Jarrod A Marto, Peter H Stone, Warren L Lee, Johan Auwerx, Peter Libby, and Mark W Feinberg. 2020. “Long noncoding RNA integrates a DNA-PK-mediated DNA damage response and vascular senescence.” Sci Transl Med, 12, 531.Abstract
Long noncoding RNAs (lncRNAs) are emerging regulators of biological processes in the vessel wall; however, their role in atherosclerosis remains poorly defined. We used RNA sequencing to profile lncRNAs derived specifically from the aortic intima of mice on a high-cholesterol diet during lesion progression and regression phases. We found that the evolutionarily conserved lncRNA small nucleolar host gene-12 () is highly expressed in the vascular endothelium and decreases during lesion progression. knockdown accelerated atherosclerotic lesion formation by 2.4-fold in mice by increased DNA damage and senescence in the vascular endothelium, independent of effects on lipid profile or vessel wall inflammation. Conversely, intravenous delivery of protected the tunica intima from DNA damage and atherosclerosis. LncRNA pulldown in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that interacted with DNA-dependent protein kinase (DNA-PK), an important regulator of the DNA damage response. The absence of reduced the DNA-PK interaction with its binding partners Ku70 and Ku80, abrogating DNA damage repair. Moreover, the anti-DNA damage agent nicotinamide riboside (NR), a clinical-grade small-molecule activator of NAD, fully rescued the increases in lesional DNA damage, senescence, and atherosclerosis mediated by knockdown. expression was also reduced in pig and human atherosclerotic specimens and correlated inversely with DNA damage and senescent markers. These findings reveal a role for this lncRNA in regulating DNA damage repair in the vessel wall and may have implications for chronic vascular disease states and aging.
Congli Zeng, Gabriel C Motta-Ribeiro, Takuga Hinoshita, Marcos Adriano Lessa, Tilo Winkler, Kira Grogg, Nathan M Kingston, John N Hutchinson, Lynette Marie Sholl, and Xiangming Fang. 2020. “Lung atelectasis promotes immune and barrier dysfunction as revealed by transcriptome sequencing in female sheep.” Anesthesiology, 133, 5, Pp. 1060-1076.
Zaniar Ghazizadeh, Tuomas Kiviniemi, Sigurast Olafsson, David Plotnick, Manu E Beerens, Kun Zhang, Leah Gillon, Michael J Steinbaugh, Victor Barrera, Shannan Ho Sui, Andreas A Werdich, Sunil Kapur, Antti Eranti, Jarmo Gunn, Juho Jalkanen, Juhani Airaksinen, Andre G Kleber, Maija Hollmén, and Calum A MacRae. 2020. “Metastable Atrial State Underlies the Primary Genetic Substrate for MYL4 Mutation-Associated Atrial Fibrillation.” Circulation, 141, 4, Pp. 301-312.Abstract
BACKGROUND: Atrial fibrillation (AF) is the most common clinical arrhythmia and is associated with heart failure, stroke, and increased mortality. The myocardial substrate for AF is poorly understood because of limited access to primary human tissue and mechanistic questions around existing in vitro or in vivo models. METHODS: Using an knock-in reporter line, we developed a protocol to generate and highly purify human pluripotent stem cell-derived cardiomyocytes displaying physiological and molecular characteristics of atrial cells. We modeled human mutants, one of the few definitive genetic causes of AF. To explore non-cell-autonomous components of AF substrate, we also created a zebrafish knockout model, which exhibited molecular, cellular, and physiologic abnormalities that parallel those in humans bearing the cognate mutations. RESULTS: There was evidence of increased retinoic acid signaling in both human embryonic stem cells and zebrafish mutant models, as well as abnormal expression and localization of cytoskeletal proteins, and loss of intracellular nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide + hydrogen. To identify potentially druggable proximate mechanisms, we performed a chemical suppressor screen integrating multiple human cellular and zebrafish in vivo endpoints. This screen identified Cx43 (connexin 43) hemichannel blockade as a robust suppressor of the abnormal phenotypes in both models of MYL4 (myosin light chain 4)-related atrial cardiomyopathy. Immunofluorescence and coimmunoprecipitation studies revealed an interaction between MYL4 and Cx43 with altered localization of Cx43 hemichannels to the lateral membrane in mutants, as well as in atrial biopsies from unselected forms of human AF. The membrane fraction from MYL4-/- human embryonic stem cell derived atrial cells demonstrated increased phospho-Cx43, which was further accentuated by retinoic acid treatment and by the presence of risk alleles at the Pitx2 locus. PKC (protein kinase C) was induced by retinoic acid, and PKC inhibition also rescued the abnormal phenotypes in the atrial cardiomyopathy models. CONCLUSIONS: These data establish a mechanistic link between the transcriptional, metabolic and electrical pathways previously implicated in AF substrate and suggest novel avenues for the prevention or therapy of this common arrhythmia.
Antonella FM Dost, Aaron L Moye, Marall Vedaie, Linh M Tran, Eileen Fung, Dar Heinze, Carlos Villacorta-Martin, Jessie Huang, Ryan Hekman, Julian H Kwan, Benjamin C Blum, Sharon M Louie, Samuel P Rowbotham, Julio Sainz de Aja, Mary E Piper, Preetida J Bhetariya, Roderick T Bronson, Andrew Emili, Gustavo Mostoslavsky, Gregory A Fishbein, William D Wallace, Kostyantyn Krysan, Steven M Dubinett, Jane Yanagawa, Darrell N Kotton, and Carla F Kim. 2020. “Organoids Model Transcriptional Hallmarks of Oncogenic KRAS Activation in Lung Epithelial Progenitor Cells.” Cell Stem Cell, 27, 4, Pp. 663-678.e8.Abstract
Mutant KRAS is a common driver in epithelial cancers. Nevertheless, molecular changes occurring early after activation of oncogenic KRAS in epithelial cells remain poorly understood. We compared transcriptional changes at single-cell resolution after KRAS activation in four sample sets. In addition to patient samples and genetically engineered mouse models, we developed organoid systems from primary mouse and human induced pluripotent stem cell-derived lung epithelial cells to model early-stage lung adenocarcinoma. In all four settings, alveolar epithelial progenitor (AT2) cells expressing oncogenic KRAS had reduced expression of mature lineage identity genes. These findings demonstrate the utility of our in vitro organoid approaches for uncovering the early consequences of oncogenic KRAS expression. This resource provides an extensive collection of datasets and describes organoid tools to study the transcriptional and proteomic changes that distinguish normal epithelial progenitor cells from early-stage lung cancer, facilitating the search for targets for KRAS-driven tumors.
Arpan C Ghosh, Sudhir Gopal Tattikota, Yifang Liu, Aram Comjean, Yanhui Hu, Victor Barrera, Shannan J Ho Sui, and Norbert Perrimon. 2020. “ PDGF/VEGF signaling from muscles to hepatocyte-like cells protects against obesity.” Elife, 9.Abstract
PDGF/VEGF ligands regulate a plethora of biological processes in multicellular organisms via autocrine, paracrine, and endocrine mechanisms. We investigated organ-specific metabolic roles of PDGF/VEGF-like factors (Pvfs). We combine genetic approaches and single-nuclei sequencing to demonstrate that muscle-derived Pvf1 signals to the hepatocyte-like cells/oenocytes to suppress lipid synthesis by activating the Pi3K/Akt1/TOR signaling cascade in the oenocytes. Functionally, this signaling axis regulates expansion of adipose tissue lipid stores in newly eclosed flies. Flies emerge after pupation with limited adipose tissue lipid stores and lipid level is progressively accumulated via lipid synthesis. We find that adult muscle-specific expression of increases rapidly during this stage and that muscle-to-oenocyte Pvf1 signaling inhibits expansion of adipose tissue lipid stores as the process reaches completion. Our findings provide the first evidence in a metazoan of a PDGF/VEGF ligand acting as a myokine that regulates systemic lipid homeostasis by activating TOR in hepatocyte-like cells.
Daniela M Santos, Lorena Pantano, Gina Pronzati, Paula Grasberger, Clemens K Probst, Katharine E Black, Jillian J Spinney, Lida P Hariri, Royale Nichols, Yufei Lin, Michael Bieler, Peter Seither, Paul Nicklin, David Wyatt, Andrew M Tager, and Benjamin D Medoff. 2020. “Screening for YAP Inhibitors Identifies Statins as Modulators of Fibrosis.” Am J Respir Cell Mol Biol, 62, 4, Pp. 479-492.Abstract
Idiopathic pulmonary fibrosis is a lung disease with limited therapeutic options that is characterized by pathological fibroblast activation and aberrant lung remodeling with scar formation. YAP (Yes-associated protein) is a transcriptional coactivator that mediates mechanical and biochemical signals controlling fibroblast activation. In this study, we developed a high-throughput small-molecule screen for YAP inhibitors in primary human lung fibroblasts. Multiple HMG-CoA (hydroxymethylglutaryl-coenzyme A) reductase inhibitors (statins) were found to inhibit YAP nuclear localization via induction of YAP phosphorylation, cytoplasmic retention, and degradation. We further show that the mevalonate pathway regulates YAP activation, and that simvastatin treatment reduces fibrosis markers in activated human lung fibroblasts and in the bleomycin mouse model of pulmonary fibrosis. Finally, we show that simvastatin modulates YAP in mouse lung fibroblasts. Our results highlight the potential of small-molecule screens for YAP inhibitors and provide a mechanism for the antifibrotic activity of statins in idiopathic pulmonary fibrosis.
Sudhir Gopal Tattikota, Bumsik Cho, Yifang Liu, Yanhui Hu, Victor Barrera, Michael J Steinbaugh, Sang-Ho Yoon, Aram Comjean, Fangge Li, Franz Dervis, Ruei-Jiun Hung, Jin-Wu Nam, Shannan Ho Sui, Jiwon Shim, and Norbert Perrimon. 2020. “A single-cell survey of blood.” Elife, 9.Abstract
blood cells, called hemocytes, are classified into plasmatocytes, crystal cells, and lamellocytes based on the expression of a few marker genes and cell morphologies, which are inadequate to classify the complete hemocyte repertoire. Here, we used single-cell RNA sequencing (scRNA-seq) to map hemocytes across different inflammatory conditions in larvae. We resolved plasmatocytes into different states based on the expression of genes involved in cell cycle, antimicrobial response, and metabolism together with the identification of intermediate states. Further, we discovered rare subsets within crystal cells and lamellocytes that express fibroblast growth factor (FGF) ligand and receptor , respectively. We demonstrate that these FGF components are required for mediating effective immune responses against parasitoid wasp eggs, highlighting a novel role for FGF signaling in inter-hemocyte crosstalk. Our scRNA-seq analysis reveals the diversity of hemocytes and provides a rich resource of gene expression profiles for a systems-level understanding of their functions.
Sarah Alsamman, Stephanie A Christenson, Amy Yu, Nadia ME Ayad, Meghan S Mooring, Joe M Segal, Jimmy Kuang-Hsien Hu, Johanna R Schaub, Steve S Ho, Vikram Rao, Megan M Marlow, Scott M Turner, Mai Sedki, Lorena Pantano, Sarani Ghoshal, Diego Dos Santos Ferreira, Hsiao-Yen Ma, Caroline C Duwaerts, Regina Espanol-Suner, Lan Wei, Benjamin Newcomb, Izolda Mileva, Daniel Canals, Yusuf A Hannun, Raymond T Chung, Aras N Mattis, Bryan C Fuchs, Andrew M Tager, Dean Yimlamai, Valerie M Weaver, Alan C Mullen, Dean Sheppard, and Jennifer Y Chen. 2020. “Targeting acid ceramidase inhibits YAP/TAZ signaling to reduce fibrosis in mice.” Sci Transl Med, 12, 557.Abstract
Hepatic stellate cells (HSCs) drive hepatic fibrosis. Therapies that inactivate HSCs have clinical potential as antifibrotic agents. We previously identified acid ceramidase (aCDase) as an antifibrotic target. We showed that tricyclic antidepressants (TCAs) reduce hepatic fibrosis by inhibiting aCDase and increasing the bioactive sphingolipid ceramide. We now demonstrate that targeting aCDase inhibits YAP/TAZ activity by potentiating its phosphorylation-mediated proteasomal degradation via the ubiquitin ligase adaptor protein β-TrCP. In mouse models of fibrosis, pharmacologic inhibition of aCDase or genetic knockout of aCDase in HSCs reduces fibrosis, stromal stiffness, and YAP/TAZ activity. In patients with advanced fibrosis, aCDase expression in HSCs is increased. Consistently, a signature of the genes most down-regulated by ceramide identifies patients with advanced fibrosis who could benefit from aCDase targeting. The findings implicate ceramide as a critical regulator of YAP/TAZ signaling and HSC activation and highlight aCDase as a therapeutic target for the treatment of fibrosis.
Lauren A Henderson, Kacie J Hoyt, Pui Y Lee, Deepak A Rao, Helena A Jonsson, Jennifer P Nguyen, Kayleigh Rutherford, Amélie M Julé, Louis-Marie Charbonnier, Siobhan Case, Margaret H Chang, Ezra M Cohen, Fatma Dedeoglu, Robert C Fuhlbrigge, Olha Halyabar, Melissa M Hazen, Erin Janssen, Susan Kim, Jeffrey Lo, Mindy S Lo, Esra Meidan, Mary Beth F Son, Robert P Sundel, Matthew L Stoll, Chad Nusbaum, James A Lederer, Talal A Chatila, and Peter A Nigrovic. 2020. “Th17 reprogramming of T cells in systemic juvenile idiopathic arthritis.” JCI Insight, 5, 6.Abstract
Systemic juvenile idiopathic arthritis (sJIA) begins with fever, rash, and high-grade systemic inflammation but commonly progresses to a persistent afebrile arthritis. The basis for this transition is unknown. To evaluate a role for lymphocyte polarization, we characterized T cells from patients with acute and chronic sJIA using flow cytometry, mass cytometry, and RNA sequencing. Acute and chronic sJIA each featured an expanded population of activated Tregs uncommon in healthy controls or in children with nonsystemic JIA. In acute sJIA, Tregs expressed IL-17A and a gene expression signature reflecting Th17 polarization. In chronic sJIA, the Th17 transcriptional signature was identified in T effector cells (Teffs), although expression of IL-17A at the protein level remained rare. Th17 polarization was abrogated in patients responding to IL-1 blockade. These findings identify evolving Th17 polarization in sJIA that begins in Tregs and progresses to Teffs, likely reflecting the impact of the cytokine milieu and consistent with a biphasic model of disease pathogenesis. The results support T cells as a potential treatment target in sJIA.
Connie WY Ha, Anthony Martin, Gregory D Sepich-Poore, Baochen Shi, Yizhou Wang, Kenneth Gouin, Gregory Humphrey, Karenina Sanders, Yasiru Ratnayake, Kelvin SL Chan, Gustaf Hendrick, JR Caldera, Christian Arias, Jacob E Moskowitz, Shannan J Ho Sui, Shaohong Yang, David Underhill, Matthew J Brady, Simon Knott, Kelly Kaihara, Michael J Steinbaugh, Huiying Li, Dermot PB McGovern, Rob Knight, Phillip Fleshner, and Suzanne Devkota. 2020. “Translocation of Viable Gut Microbiota to Mesenteric Adipose Drives Formation of Creeping Fat in Humans.” Cell, 183, 3, Pp. 666-683.e17.Abstract
A mysterious feature of Crohn's disease (CD) is the extra-intestinal manifestation of "creeping fat" (CrF), defined as expansion of mesenteric adipose tissue around the inflamed and fibrotic intestine. In the current study, we explore whether microbial translocation in CD serves as a central cue for CrF development. We discovered a subset of mucosal-associated gut bacteria that consistently translocated and remained viable in CrF in CD ileal surgical resections, and identified Clostridium innocuum as a signature of this consortium with strain variation between mucosal and adipose isolates, suggesting preference for lipid-rich environments. Single-cell RNA sequencing characterized CrF as both pro-fibrotic and pro-adipogenic with a rich milieu of activated immune cells responding to microbial stimuli, which we confirm in gnotobiotic mice colonized with C. innocuum. Ex vivo validation of expression patterns suggests C. innocuum stimulates tissue remodeling via M2 macrophages, leading to an adipose tissue barrier that serves to prevent systemic dissemination of bacteria.
Sining Leng, Emanuele Pignatti, Radhika S Khetani, Manasvi S Shah, Simiao Xu, Ji Miao, Makoto M Taketo, Felix Beuschlein, Paula Q Barrett, Diana L Carlone, and David T Breault. 2020. “β-Catenin and FGFR2 regulate postnatal rosette-based adrenocortical morphogenesis.” Nat Commun, 11, 1, Pp. 1680.Abstract
Rosettes are widely used in epithelial morphogenesis during embryonic development and organogenesis. However, their role in postnatal development and adult tissue maintenance remains largely unknown. Here, we show zona glomerulosa cells in the adult adrenal cortex organize into rosettes through adherens junction-mediated constriction, and that rosette formation underlies the maturation of adrenal glomerular structure postnatally. Using genetic mouse models, we show loss of β-catenin results in disrupted adherens junctions, reduced rosette number, and dysmorphic glomeruli, whereas β-catenin stabilization leads to increased adherens junction abundance, more rosettes, and glomerular expansion. Furthermore, we uncover numerous known regulators of epithelial morphogenesis enriched in β-catenin-stabilized adrenals. Among these genes, we show Fgfr2 is required for adrenal rosette formation by regulating adherens junction abundance and aggregation. Together, our data provide an example of rosette-mediated postnatal tissue morphogenesis and a framework for studying the role of rosettes in adult zona glomerulosa tissue maintenance and function.
Joseph R Klim, Luis A Williams, Francesco Limone, Irune Guerra San Juan, Brandi N Davis-Dusenbery, Daniel A Mordes, Aaron Burberry, Michael J Steinbaugh, Kanchana K Gamage, Rory Kirchner, Rob Moccia, Seth H Cassel, Kuchuan Chen, Brian J Wainger, Clifford J Woolf, and Kevin Eggan. 2019. “ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair.” Nat Neurosci, 22, 2, Pp. 167-179.Abstract
The findings that amyotrophic lateral sclerosis (ALS) patients almost universally display pathological mislocalization of the RNA-binding protein TDP-43 and that mutations in its gene cause familial ALS have nominated altered RNA metabolism as a disease mechanism. However, the RNAs regulated by TDP-43 in motor neurons and their connection to neuropathy remain to be identified. Here we report transcripts whose abundances in human motor neurons are sensitive to TDP-43 depletion. Notably, expression of STMN2, which encodes a microtubule regulator, declined after TDP-43 knockdown and TDP-43 mislocalization as well as in patient-specific motor neurons and postmortem patient spinal cord. STMN2 loss upon reduced TDP-43 function was due to altered splicing, which is functionally important, as we show STMN2 is necessary for normal axonal outgrowth and regeneration. Notably, post-translational stabilization of STMN2 rescued neurite outgrowth and axon regeneration deficits induced by TDP-43 depletion. We propose that restoring STMN2 expression warrants examination as a therapeutic strategy for ALS.
Peter Krusche, Len Trigg, Paul C Boutros, Christopher E Mason, Francisco M De La Vega, Benjamin L Moore, Mar Gonzalez-Porta, Michael A Eberle, Zivana Tezak, Samir Lababidi, Rebecca Truty, George Asimenos, Birgit Funke, Mark Fleharty, Brad A Chapman, Marc Salit, and Justin M Zook. 2019. “Best practices for benchmarking germline small-variant calls in human genomes.” Nat Biotechnol, 37, 5, Pp. 555-560.Abstract
Standardized benchmarking approaches are required to assess the accuracy of variants called from sequence data. Although variant-calling tools and the metrics used to assess their performance continue to improve, important challenges remain. Here, as part of the Global Alliance for Genomics and Health (GA4GH), we present a benchmarking framework for variant calling. We provide guidance on how to match variant calls with different representations, define standard performance metrics, and stratify performance by variant type and genome context. We describe limitations of high-confidence calls and regions that can be used as truth sets (for example, single-nucleotide variant concordance of two methods is 99.7% inside versus 76.5% outside high-confidence regions). Our web-based app enables comparison of variant calls against truth sets to obtain a standardized performance report. Our approach has been piloted in the PrecisionFDA variant-calling challenges to identify the best-in-class variant-calling methods within high-confidence regions. Finally, we recommend a set of best practices for using our tools and evaluating the results.
Nicco Krezdorn, Christine G Lian, Michael Wells, Luccie Wo, Sotirios Tasigiorgos, Shuyun Xu, Thiago J Borges, Rayven M Frierson, Ewelina Stanek, Leonardo V Riella, Bohdan Pomahac, and George F Murphy. 2019. “Chronic rejection of human face allografts.” Am J Transplant, 19, 4, Pp. 1168-1177.Abstract
Face vascularized composite allografts (FVCAs) have helped patients with severe facial disfigurement, with acute rejection now largely controlled through iatrogenic immunosuppression. However, little is known regarding the incidence and mechanism(s) of more long-term pathologic alterations in FVCAs that may affect function and graft durability. Protocol surveillance biopsy specimens for up to an 8-year interval in 7 patients who received FVCAs at our institution revealed histopathologic evidence of chronic rejection. Clinical manifestations included features of premature aging, mottled leukoderma accentuating suture lines, telangiectasia, and dryness of nasal mucosa. Pathologic changes consisted of epidermal thinning accompanied by discrete foci of lymphocyte-mediated cytotoxicity, hyperkeratosis, follicular plugging, vascular ectasia, and sclerosis beneath the epidermal layer associated with collagen type I deposition. Genomic interrogation and immunohistochemistry of sclerotic zones revealed upregulation of the AP-1 pathway components, JunB and c-Fos, previously implicated in overproduction of type I dermal collagen in the setting of systemic sclerosis. We conclude that some patients develop chronic rejection in FVCAs with striking similarities to alterations seen in certain autoimmune cutaneous disorders (lupus erythematosus and scleroderma/chronic sclerodermoid graft-versus-host disease). Identification of relevant pathways and genes, such as JunB and c-Fos, may provide new targets for preventative therapies for chronic immune-mediated changes in vascularized composite allografts.