• BIG-TREE: Base-Edited Isogenic hPSC Line Generation Using a Transient Reporter for Editing Enrichment

      Brookhouser, Nicholas; Tekel, Stefan J; Standage-Beier, Kylie; Nguyen, Toan; Schwarz, Grace; Wang, Xiao; Brafman, David A; Univ Arizona, Coll Med Phoenix, Grad Program Clin Translat Sci (CELL PRESS, 2020-01-30)
      Current CRISPR-targeted single-nucleotide modifications and subsequent isogenic cell line generation in human pluripotent stem cells (hPSCs) require the introduction of deleterious double-stranded DNA breaks followed by inefficient homology-directed repair (HDR). Here, we utilize Cas9 deaminase base-editing technologies to co-target genomic loci and an episomal reporter to enable single-nucleotide genomic changes in hPSCs without HDR. Together, this method entitled base-edited isogenic hPSC line generation using a transient reporter for editing enrichment (BIG-TREE) allows for single-nucleotide editing efficiencies of >80% across multiple hPSC lines. In addition, we show that BIG-TREE allows for efficient generation of loss-of-function hPSC lines via introduction of premature stop codons. Finally, we use BIG-TREE to achieve efficient multiplex editing of hPSCs at several independent loci. This easily adoptable method will allow for the precise and efficient base editing of hPSCs for use in developmental biology, disease modeling, drug screening, and cell-based therapies.
    • Category Selectivity for Face and Scene Recognition in Human Medial Parietal Cortex

      Woolnough, Oscar; Rollo, Patrick S; Forseth, Kiefer J; Kadipasaoglu, Cihan M; Ekstrom, Arne D; Tandon, Nitin; Univ Arizona, Dept Psychol (CELL PRESS, 2020-06-04)
      The rapid recognition and memory of faces and scenes implies the engagement of category-specific computational hubs in the ventral visual stream with the distributed cortical memory network. To better understand how recognition and identification occur in humans, we performed direct intracranial recordings, in a large cohort of patients (n = 50), from the medial parietal cortex (MPC) and the medial temporal lobe (MTL), structures known to be engaged during face and scene identification. We discovered that the MPC is topologically tuned to face and scene recognition, with clusters in MPC performing scene recognition bilaterally and face recognition in right subparietal sulcus. The MTL displayed a selectivity gradient with anterior, entorhinal cortex showing face selectivity and posterior parahippocampal regions showing scene selectivity. In both MPC and MTL, stimulus-specific identifiable exemplars led to greater activity in these cortical patches. These two regions work in concert for recognition of faces and scenes. Feature selectivity and identity-sensitive activity in the two regions was coincident, and they exhibited theta-phase locking during face and scene recognition. These findings together provide clear evidence for a specific role of subregions in the MPC for the recognition of unique entities.
    • Cellular Contraction Can Drive Rapid Epithelial Flows

      Vig, Dhruv K; Hamby, Alex E; Wolgemuth, Charles W; Univ Arizona, Dept Mol & Cellular Biol; Univ Arizona, Dept Phys (CELL PRESS, 2017-10-03)
      Single, isolated epithelial cells move randomly; however, during wound healing, organism development, cancer metastasis, and many other multicellular phenomena, motile cells group into a collective and migrate persistently in a directed manner. Recent work has examined the physics and biochemistry that coordinates the motions of these groups of cells. Of late, two mechanisms have been touted as being crucial to the physics of these systems: leader cells and jamming. However, the actual importance of these to collective migration remains circumstantial. Fundamentally, collective behavior must arise from the actions of individual cells. Here, we show how biophysical activity of an isolated cell impacts collective dynamics in epithelial layers. Although many reports suggest that wound closure rates depend on isolated cell speed and/or leader cells, we find that these correlations are not universally true, nor do collective dynamics follow the trends suggested by models for jamming. Instead, our experimental data, when coupled with a mathematical model for collective migration, shows that intracellular contractile stress, isolated cell speed, and adhesion all play a substantial role in influencing epithelial dynamics, and that alterations in contraction and/or substrate adhesion can cause confluent epithelial monolayers to exhibit an increase in motility, a feature reminiscent of cancer metastasis. These results directly question the validity of wound-healing assays as a general means for measuring cell migration, and provide further insight into the salient physics of collective migration.
    • Changing Climate Drives Divergent and Nonlinear Shifts in Flowering Phenology across Elevations

      Rafferty, Nicole E; Diez, Jeffrey M; Bertelsen, C David; Univ Arizona, Sch Nat Resources & Environm; Univ Arizona, Herbarium (CELL PRESS, 2020-02-03)
      Climate change is known to affect regional weather patterns and phenology; however, we lack understanding of how climate drives phenological change across local spatial gradients. This spatial variation is critical for determining whether subpopulations and metacommunities are changing in unison or diverging in phenology. Divergent responses could reduce synchrony both within species (disrupting gene flow among subpopulations) and among species (disrupting interspecific interactions in communities). We also lack understanding of phenological change in environments where life history events are frequently aseasonal, such as the tropical, arid, and semi-arid ecosystems that cover vast areas. Using a 33-year-long dataset spanning a 1,267-m semi-arid elevational gradient in the southwestern United States, we test whether flowering phenology diverged among subpopulations within species and among five communities comprising 590 species. Applying circular statistics to test for changes in year-round flowering, we show flowering has become earlier for all communities except at the highest elevations. However, flowering times shifted at different rates across elevations likely because of elevation-specific changes in temperature and precipitation, indicating diverging phenologies of neighboring communities. Subpopulations of individual species also diverged at mid-elevation but converged in phenology at high elevation. These changes in flowering phenology among communities and subpopulations are undetectable when data are pooled across the gradient. Furthermore, we show that nonlinear changes in flowering times over the 33-year record are obscured by traditional calculations of long-term trends. These findings reveal greater spatiotemporal complexity in phenological responses than previously recognized and indicate climate is driving phenological reshuffling across local spatial gradients.
    • Controlling Depth of Cellular Quiescence by an Rb-E2F Network Switch

      Kwon, Jungeun Sarah; Everetts, Nicholas J.; Wang, Xia; Wang, Weikang; Della Croce, Kimiko; Xing, Jianhua; Yao, Guang; Univ Arizona, Dept Mol & Cellular Biol; Univ Arizona, Arizona Canc Ctr (CELL PRESS, 2017-09)
      Quiescence is a non-proliferative cellular state that is critical to tissue repair and regeneration. Although often described as the G0 phase, quiescence is not a single homogeneous state. As cells remain quiescent for longer durations, they move progressively deeper and display a reduced sensitivity to growth signals. Deep quiescent cells, unlike senescent cells, can still re-enter the cell cycle under physiological conditions. Mechanisms controlling quiescence depth are poorly understood, representing a currently underappreciated layer of complexity in growth control. Here, we show that the activation threshold of a Retinoblastoma (Rb)-E2F network switch controls quiescence depth. Particularly, deeper quiescent cells feature a higher E2F-switching threshold and exhibit a delayed traverse through the restriction point (R-point). We further show that different components of the Rb-E2F network can be experimentally perturbed, following computer model predictions, to coarse-or fine-tune the E2F-switching threshold and drive cells into varying quiescence depths.
    • The Dark Side of the Genome: Revealing the Native Transposable Element/Repeat Content of Eukaryotic Genomes

      Copetti, Dario; Wing, Rod A.; Arizona Genomics Institute, BIO5 Institute and School of Plant Sciences (CELL PRESS, 2016-09)
      The majority of genome assemblies to date fail to represent the true structure of native genomes. This lack of completeness is largely due to the inability to assemble the variable (often significant) fraction of nuclear genomes that is composed primarily of repeated sequences (with either a structural function such as satellite DNA and simple sequence repeats or “selfish DNA” such as high-copy transposable elements [TEs]), herein defined as the “dark side of the genome.” To address this problem, we developed a method to detect and quantify the dark side of the genome and used it to infer the genomic composition and dynamic evolution of the majority of native repeats and TEs present within several test eukaryotic genomes.
    • Defects in Antiviral T Cell Responses Inflicted by Aging-Associated miR-181a Deficiency

      Kim, Chulwoo; Jadhav, Rohit R; Gustafson, Claire E; Smithey, Megan J; Hirsch, Alec J; Uhrlaub, Jennifer L; Hildebrand, William H; Nikolich-Žugich, Janko; Weyand, Cornelia M; Goronzy, Jörg J; et al. (CELL PRESS, 2019-11-19)
      Generation of protective immunity to infections and vaccinations declines with age. Studies in healthy individuals have implicated reduced miR-181a expression in T cells as contributing to this defect. To understand the impact of miR-181a expression on antiviral responses, we examined LCMV infection in mice with miR-181ab1-deficient T cells. We found that miR-181a deficiency delays viral clearance, thereby biasing the immune response in favor of CD4 over CD8 T cells. Antigen-specific CD4 T cells in mice with miR-181a-deficient T cells expand more and have a broader TCR repertoire with preferential expansion of high-affinity T cells than in wild-type mice. Importantly, generation of antigen-specific miR-181a-deficient CD8 effector T cells is particularly impaired, resulting in lower frequencies of CD8 T cells in the liver even at time points when the infection has been cleared. Consistent with the mouse model, CD4 memory T cells in individuals infected with West Nile virus at older ages tend to be more frequent and of higher affinity.
    • Distinct Circuits for Recovery of Eye Dominance and Acuity in Murine Amblyopia

      Stephany, Céleste-Élise; Ma, Xiaokuang; Dorton, Hilary M.; Wu, Jie; Solomon, Alexander M.; Frantz, Michael G.; Qiu, Shenfeng; McGee, Aaron W.; Univ Arizona, Dept Basic Med Sci, Coll Med (CELL PRESS, 2018-06-18)
      Degrading vision by one eye during a developmental critical period yields enduring deficits in both eye dominance and visual acuity. A predominant model is that "reactivating'' ocular dominance (OD) plasticity after the critical period is required to improve acuity in amblyopic adults. However, here we demonstrate that plasticity of eye dominance and acuity are independent and restricted by the nogo-66 receptor (ngr1) in distinct neuronal populations. Ngr1 mutant mice display greater excitatory synaptic input onto both inhibitory and excitatory neurons with restoration of normal vision. Deleting ngr1 in excitatory cortical neurons permits recovery of eye dominance but not acuity. Reciprocally, deleting ngr1 in thalamus is insufficient to rectify eye dominance but yields improvement of acuity to normal. Abolishing ngr1 expression in adult mice also promotes recovery of acuity. Together, these findings challenge the notion that mechanisms for OD plasticity contribute to the alterations in circuitry that restore acuity in amblyopia.
    • Immune Monitoring Reveals Fusion Peptide Priming to Imprint Cross-Clade HIV-Neutralizing Responses with a Characteristic Early B Cell Signature

      Cheng, Cheng; Duan, Hongying; Xu, Kai; Chuang, Gwo-Yu; Corrigan, Angela R.; Geng, Hui; O'Dell, Sijy; Ou, Li; Chambers, Michael; Changela, Anita; et al. (CELL PRESS, 2020-08)
      The HIV fusion peptide (FP) is a promising vaccine target. FP-directed monoclonal antibodies from vaccinated macaques have been identified that neutralize up to similar to 60% of HIV strains; these vaccinations, however, have involved similar to 1 year with an extended neutralization-eclipse phase without measurable serum neutralization. Here, in 32 macaques, we test seven vaccination regimens, each comprising multiple immunizations of FP-carrier conjugates and HIV envelope (Env) trimers. Comparisons of vaccine regimens reveal FP-carrier conjugates to imprint cross-clade neutralizing responses and a cocktail of FP conjugate and Env trimer to elicit the earliest broad responses. We identify a signature, appearing as early as week 6 and involving the frequency of B cells recognizing both FP and Env trimer, predictive of vaccine-elicited breadth similar to 1 year later. Immune monitoring of B cells in response to vaccination can thus enable vaccine insights even in the absence of serum neutralization, here identifying FP imprinting, cocktail approach, and early signature as means to improve FP-directed vaccine responses.
    • Integrated Patient-Derived Models Delineate Individualized Therapeutic Vulnerabilities of Pancreatic Cancer.

      Witkiewicz, Agnieszka K; Balaji, Uthra; Eslinger, Cody; McMillan, Elizabeth; Conway, William; Posner, Bruce; Mills, Gordon B; O'Reilly, Eileen M; Knudsen, Erik S; Univ Arizona, Dept Pathol; et al. (CELL PRESS, 2016-08-16)
      Pancreatic ductal adenocarcinoma (PDAC) harbors the worst prognosis of any common solid tumor, and multiple failed clinical trials indicate therapeutic recalcitrance. Here, we use exome sequencing of patient tumors and find multiple conserved genetic alterations. However, the majority of tumors exhibit no clearly defined therapeutic target. High-throughput drug screens using patient-derived cell lines found rare examples of sensitivity to monotherapy, with most models requiring combination therapy. Using PDX models, we confirmed the effectiveness and selectivity of the identified treatment responses. Out of more than 500 single and combination drug regimens tested, no single treatment was effective for the majority of PDAC tumors, and each case had unique sensitivity profiles that could not be predicted using genetic analyses. These data indicate a shortcoming of reliance on genetic analysis to predict efficacy of currently available agents against PDAC and suggest that sensitivity profiling of patient-derived models could inform personalized therapy design for PDAC.
    • Mechanism of Filamentation-Induced Allosteric Activation of the SgrAI Endonuclease

      Polley, Smarajit; Lyumkis, Dmitry; Horton, Nancy C; Univ Arizona, Dept Mol & Cellular Biol (CELL PRESS, 2019-10-01)
      Filament formation by enzymes is increasingly recognized as an important phenomenon with potentially unique regulatory properties and biological roles. SgrAI is an allosterically regulated type II restriction endonuclease that forms filaments with enhanced DNA cleavage activity and altered sequence specificity. Here, we present the cryoelectron microscopy (cryo-EM) structure of the filament of SgrAI in its activated configuration. The structural data illuminate the mechanistic origin of hyperaccelerated DNA cleavage activity and suggests how indirect DNA sequence readout within filamentous SgrAI may enable recognition of substantially more nucleotide sequences than its low-activity form, thereby altering and partially relaxing its DNA sequence specificity. Together, substrate DNA binding, indirect readout, and filamentation simultaneously enhance SgrAI's catalytic activity and modulate substrate preference. This unusual enzyme mechanism may have evolved to perform the specialized functions of bacterial innate immunity in rapid defense against invading phage DNA without causing damage to the host DNA.
    • Metabolic and Transcriptional Modules Independently Diversify Plasma Cell Lifespan and Function

      Lam, Wing Y.; Jash, Arijita; Yao, Cong-Hui; D’Souza, Lucas; Wong, Rachel; Nunley, Ryan M.; Meares, Gordon P.; Patti, Gary J.; Bhattacharya, Deepta; Univ Arizona, Coll Med, Dept Immunobiol (CELL PRESS, 2018-08-28)
      Plasma cell survival and the consequent duration of immunity vary widely with infection or vaccination. Using fluorescent glucose analog uptake, we defined multiple developmentally independent mouse plasma cell populations with varying lifespans. Long-lived plasma cells imported more fluorescent glucose analog, expressed higher surface levels of the amino acid transporter CD98, and had more autophagosome mass than did short-lived cells. Low amino acid concentrations triggered reductions in both antibody secretion and mitochondrial respiration, especially by short-lived plasma cells. To explain these observations, we found that glutamine was used for both mitochondrial respiration and anaplerotic reactions, yielding glutamate and aspartate for antibody synthesis. Endoplasmic reticulum (ER) stress responses, which link metabolism to transcriptional outcomes, were similar between long- and short-lived subsets. Accordingly, population and single-cell transcriptional comparisons across mouse and human plasma cell subsets revealed few consistent and conserved differences. Thus, plasma cell antibody secretion and lifespan are primarily defined by non-transcriptional metabolic traits.
    • A Modality-Independent Network Underlies the Retrieval of Large-Scale Spatial Environments in the Human Brain

      Huffman, Derek J; Ekstrom, Arne D; Univ Arizona, Psychol Dept (CELL PRESS, 2019-11-06)
      In humans, the extent to which body-based cues, such as vestibular, somatosensory, and motoric cues, are necessary for normal expression of spatial representations remains unclear. Recent breakthroughs in immersive virtual reality technology allowed us to test how body-based cues influence spatial representations of large-scale environments in humans. Specifically, we manipulated the availability of body-based cues during navigation using an omnidirectional treadmill and a head-mounted display, investigating brain differences in levels of activation (i.e., univariate analysis), patterns of activity (i.e., multivariate pattern analysis), and putative network interactions between spatial retrieval tasks using fMRI. Our behavioral and neuroimaging results support the idea that there is a core, modality-independent network supporting spatial memory retrieval in the human brain. Thus, for well-learned spatial environments, at least in humans, primarily visual input may be sufficient for expression of complex representations of spatial environments.
    • Motivation and Engagement during Visually Guided Behavior

      Ortiz, Alexander V; Aziz, David; Hestrin, Shaul; Univ Arizona, Coll Opt Sci (CELL PRESS, 2020-10-20)
      Animal behavior is motivated by internal drives, such as thirst and hunger, generated in hypothalamic neurons that project widely to many brain areas. We find that water-restricted mice maintain stable, high-level contrast sensitivity and brief reaction time while performing a visual task, but then abruptly stop and become disengaged. Mice consume a significant amount of water when freely provided in their home cage immediately after the task, indicating that disengagement does not reflect cessation of thirst. Neuronal responses of V1 neurons are reduced in the disengaged state, but pupil diameter does not decrease, suggesting that animals' reduced level of arousal does not drive the transition to disengagement. Our findings indicate that satiation level alone does not have an instructive role in visually guided behavior and suggest that animals' behavior is governed by cost-benefit analysis that can override thirst signals.
    • Neural Circuit Mechanism Underlying the Feeding Controlled by Insula-Central Amygdala Pathway

      Zhang-Molina, Calvin; Schmit, Matthew B; Cai, Haijiang; Univ Arizona, Dept Math; Univ Arizona, Dept Neurosci; Univ Arizona, Grad Interdisciplinary Program Neurosci; Univ Arizona, Bio5 Inst; Univ Arizona, Dept Neurol (CELL PRESS, 2020-04-24)
      The Central nucleus of amygdala (CeA) contains distinct populations of neurons that play opposing roles in feeding. The circuit mechanism of how CeA neurons process information sent fromtheir upstreaminputs to regulate feeding is still unclear. Here we show that activation of the neural pathway projecting from insular cortex neurons to the CeA suppresses food intake. Surprisingly, we find that the inputs from insular cortex form excitatory connections with similar strength to all types of CeA neurons. To reconcile this puzzling result, and previous findings, we developed a conductance-based dynamical systems model for the CeA neuronal network. Computer simulations showed that both the intrinsic electrophysiological properties of individual CeA neurons and the overall synaptic organization of the CeA circuit play a functionally significant role in shaping CeA neural dynamics. We successfully identified a specific CeA circuit structure that reproduces the desired circuit output consistent with existing experimentally observed feeding behaviors.
    • Non-enzymatic Lysine Lactoylation of Glycolytic Enzymes

      Gaffney, Dominique O; Jennings, Erin Q; Anderson, Colin C; Marentette, John O; Shi, Taoda; Schou Oxvig, Anne-Mette; Streeter, Matthew D; Johannsen, Mogens; Spiegel, David A; Chapman, Eli; et al. (CELL PRESS, 2020-02-20)
      Post-translational modifications (PTMs) regulate enzyme structure and function to expand the functional proteome. Many of these PTMs are derived from cellular metabolites and serve as feedback and feedforward mechanisms of regulation. We have identified a PTM that is derived from the glycolytic by-product, methylglyoxal. This reactive metabolite is rapidly conjugated to glutathione via glyoxalase 1, generating lactoylglutathione (LGSH). LGSH is hydrolyzed by glyoxalase 2 (GLO2), cycling glutathione and generating D-lactate. We have identified the non-enzymatic acyl transfer of the lactate moiety from LGSH to protein Lys residues, generating a "LactoylLys'' modification on proteins. GLO2 knockout cells have elevated LGSH and a consequent marked increase in LactoylLys. Using an alkyne-tagged methylglyoxal analog, we show that these modifications are enriched on glycolytic enzymes and regulate glycolysis. Collectively, these data suggest a previously unexplored feedback mechanism that may serve to regulate glycolytic flux under hyperglycemic or Warburg-like conditions.
    • Plk4 Regulates Centriole Asymmetry and Spindle Orientation in Neural Stem Cells

      Gambarotto, Davide; Pennetier, Carole; Ryniawec, John M; Buster, Daniel W; Gogendeau, Delphine; Goupil, Alix; Nano, Maddalena; Simon, Anthony; Blanc, Damien; Racine, Victor; et al. (CELL PRESS, 2019-07-01)
      Defects in mitotic spindle orientation (MSO) disrupt the organization of stem cell niches impacting tissue morphogenesis and homeostasis. Mutations in centrosome genes reduce MSO fidelity, leading to tissue dysplasia and causing several diseases such as microcephaly, dwarfism, and cancer. Whether these mutations perturb spindle orientation solely by affecting astral microtubule nucleation or whether centrosome proteins have more direct functions in regulatingMSO is unknown. To investigate this question, we analyzed the consequences of deregulating Plk4 (the master centriole duplication kinase) activity in Drosophila asymmetrically dividing neural stem cells. We found that Plk4 functions upstream of MSO control, orchestrating centriole symmetry breaking and consequently centrosome positioning. Mechanistically, we show that Plk4 acts through Spd2 phosphorylation, which induces centriole release from the apical cortex. Overall, this work not only reveals a role for Plk4 in regulating centrosome function but also links the centrosome biogenesis machinery with the MSO apparatus.
    • Post-transcriptional Inhibition of Hsc70-4/HSPA8 Expression Leads to Synaptic Vesicle Cycling Defects in Multiple Models of ALS

      Coyne, Alyssa N.; Lorenzini, Ileana; Chou, Ching-Chieh; Torvund, Meaghan; Rogers, Robert S.; Starr, Alexander; Zaepfel, Benjamin L.; Levy, Jennifer; Johannesmeyer, Jeffrey; Schwartz, Jacob C.; et al. (CELL PRESS, 2017-10)
      Amyotrophic lateral sclerosis (ALS) is a synaptopathy accompanied by the presence of cytoplasmic aggregates containing TDP-43, an RNA-binding protein linked to similar to 97% of ALS cases. Using a Drosophila model of ALS, we show that TDP-43 overexpression (OE) in motor neurons results in decreased expression of the Hsc70-4 chaperone at the neuromuscular junction (NMJ). Mechanistically, mutant TDP-43 sequesters hsc70-4 mRNA and impairs its translation. Expression of the Hsc70-4 ortholog, HSPA8, is also reduced in primary motor neurons and NMJs of mice expressing mutant TDP-43. Electrophysiology, imaging, and genetic interaction experiments reveal TDP-43-dependent defects in synaptic vesicle endocytosis. These deficits can be partially restored by OE of Hsc70-4, cysteine-string protein (Csp), or dynamin. This suggests that TDP-43 toxicity results in part from impaired activity of the synaptic CSP/Hsc70 chaperone complex impacting dynamin function. Finally, Hsc70-4/HSPA8 expression is also post-transcriptionally reduced in fly and human induced pluripotent stem cell (iPSC) C9orf72 models, suggesting a common disease pathomechanism.
    • Rapid Transduction and Expansion of Transduced T Cells with Maintenance of Central Memory Populations

      Pampusch, Mary S; Haran, Kumudhini Preethi; Hart, Geoffrey T; Rakasz, Eva G; Rendahl, Aaron K; Berger, Edward A; Connick, Elizabeth; Skinner, Pamela J; Univ Arizona, Div Infect Dis (CELL PRESS, 2019-09-30)
      Chimeric antigen receptor (CAR)-T cells show great promise in treating cancers and viral infections. However, most protocols developed to expand T cells require relatively long periods of time in culture, potentially leading to progression toward populations of terminally differentiated effector memory cells. Here, we describe in detail a 9-day protocol for CAR gene transduction and expansion of primary rhesus macaque peripheral blood mononuclear cells (PBMCs). Cells produced and expanded with this method show high levels of viability, high levels of co-expression of two transduced genes, retention of the central memory phenotype, and sufficient quantity for immunotherapeutic infusion of 1-2 × 108 cells/kg in a 10 kg rhesus macaque. This 9-day protocol may be broadly used for CAR-T cell and other T cell immunotherapy approaches to decrease culture time and increase maintenance of central memory populations.
    • Rare Earth Doped Optical Fibers with Multi-section Core

      Huang, Chongyuan; Geng, Jihong; Luo, Tao; Han, Jiali; Wang, Qing; Liang, Rongguang; Fan, Shanhui; Jiang, Shibin; Univ Arizona, Coll Opt Sci; Univ Arizona, Dept Syst & Ind Engn (CELL PRESS, 2019-12-20)
      The gain bandwidth of a single-mode fiber is limited by the atomic transitions of one rare earth gain element. Here we overcome this long-standing challenge by designing a new single-mode fiber with multi-section core, where each section is doped with different gain element. We theoretically propose and experimentally demonstrate that this configuration provides a gain bandwidth well beyond the capability of conventional design, whereas the inclusion of multiple sections does not compromise single-mode operation or the quality of the transverse modal profile. This new fiber will be beneficial in realizing all fiber laser systems with few-cycle pulse duration or octave tunability.