bluuebunny/crossref_metadata_2025 huggingface dataset:
Kynurenine, a tryptophan metabolite that increases with age, induces skeletal muscle atrophy and reactive oxygen species.
Authors: Kaiser Helen , Yu Kanglun , Mendhe Bharati , Fulzele Sadanand , Johnson Maribeth , McGee‐Lawrence Meghan , Hamrick Mark W.
Published: June 2021

Loss of muscle mass and power with age in the form of sarcopenia is associated with a loss of independence and a substantial decrease in quality of life due to an increase in falls and fractures. The cellular and molecular mechanisms underlying sarcopenia are not well‐understood; however, heterochronic parabiosis experiments have revealed that circulating factors are likely to play a role, and reactive oxygen species (ROS) are also known to increase in skeletal muscle with aging. This study investigated the relationship between kynurenine, a circulating tryptophan metabolite which increases with age, and markers of muscle oxidative stress. C2C12 myoblasts were treated with kynurenine in a dose‐dependent manner (0, 1, 10 μM) and ROS was measured using an Amplex red assay. Female C57BL/6 mice 6 months of age were treated with kynurenine (10 mg/kg BW) or with saline (vehicle control) for 4 weeks. Muscle mass and fiber size were measured from the quadriceps femoris ex vivo, and ROS was measured from paraffin‐embedded muscle sections using immunostaining for 4HNE. ROS was increased 2‐fold in C2C12 myoblasts treated with kynurenine at both low and high concentrations (P<.01). Quadriceps weight relative to body weight was reduced (10%) and muscle fiber size was significantly lower (P<.01) in young mice treated with kynurenine compared to controls. ROS was increased by 20% (P<.05) in young mice treated with kynurenine compared to control based on 4HNE staining. Our data reveal that the circulating tryptophan metabolite kynurenine can induce muscle wasting and increase reactive oxygen species in skeletal muscle, suggesting that pharmacological approaches to inhibit kynurenine production may provide a therapeutic strategy for the prevention of sarcopenia.

Support or Funding Information

Funding for this research was provided by the National Institute on Aging (P01 AG036675).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Age‐induced Hepatic Steatosis and Inflammation of Murine Livers is Influenced by MCP‐1
Authors: Stahl Elizabeth , LoPresti Samuel , Delgado Evan , Alencastro Frances , Wilkinson Patrick , Duncan Andrew W. , Brown Bryan N.
Published: June 2021

Aging is linked to the onset and severity of several chronic liver diseases, including non‐alcoholic fatty liver disease, alcoholic liver disease, and hepatitis, which correlate with high levels of macrophage infiltration, inflammation, and immunosenescence. Recent findings suggest that liver macrophages can be divided into two unique subsets: CD68+ embryonically‐derived Kupffer cells and CD11b+ bone marrow derived cells, which exhibit specific cell functions. It is currently unclear how the macrophage populations residing in the liver are phenotypically and functionally impacted by the natural aging process, which may have implications for the onset and progression of inflammatory disease and liver pathology in elderly populations.

The aim of this study is to characterize the liver macrophage compartment from young (8–10 week) and aged (18–20 month) C57BL/6 wild‐type mice. First, we identified a significant increase in the total number of F4/80+ macrophages residing in aged livers. Preliminary in vitro studies showed that aged macrophages were primed towards inflammatory reaction, including increased phagocytosis and nitric oxide production, in response to LPS and interferon‐gamma. In addition, the aged macrophage population contained a greater proportion of CD11b+ cells, which are most likely derived from circulating monocytes. To confirm this, we determined that aged hepatocytes secrete enhanced levels of monocyte chemoattractant protein‐1 (MCP‐1), an important signaling molecule for monocyte recruitment following injury. Although aged livers do not show signs of injury or damage, we determined that hepatocytes from aged livers have increased triglyceride content and a shifted fatty acid profile. Of note, the aged mice received the same diet as young controls, and do not show signs of glucose intolerance. Future studies will examine how inhibition of MCP‐1 signaling will impact macrophage recruitment, inflammation, and steatosis in aged livers towards a better understanding of age‐related liver pathology.

Support or Funding Information

NIH‐NIBIB Training Grant (T32‐EB001026); NIH‐NIDDK NRSA Fellowship (F31‐DK112633‐01A1); Pittsburgh Liver Research Center Pilot & Feasibility Grant

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Postnatal Organogenesis by Transdifferentiation
Authors: Willenbring Holger
Published: June 2021

Transdifferentiation is a physiological backup mechanism and alternative to stem‐cell‐mediated organ regeneration. In adult mammals, findings of transdifferentiation have been limited to replenishing lost cells in preexisting structures, i.e., in the presence of a fully developed scaffold and niche. Here we show that hepatocytes in the adult liver can transdifferentiate to build a structure that failed to form in development––the biliary system in mice that mimic the hepatic phenotype of human Alagille syndrome. The bile ducts forming the new biliary system are fully functional as evidenced by reversal of cholestasis, liver injury and liver fibrosis. Our results reveal the potential of mammalian transdifferentiation and may inform efforts to build organs containing a complex tubular system.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phospholipid Regulation of Inflammatory Processes and Viral Infection
Authors: Voelker Dennis R.
Published: June 2021

Gas exchange between the external environment and erythrocyte hemoglobin takes place within the alveolar compartment of the lung, which contains an extracellular, phospholipid‐rich complex known as pulmonary surfactant. The major phospholipid classes of pulmonary surfactant consist of phosphatidylcholine (~70%), phosphatidylglycerol (PG) (~10%) and phosphatidylinositol (PI) (~2%). Dipalmitoyl‐phosphatidylcholine plays an essential role in reducing surface tension within the alveoli during respiration, but the roles played by phosphatidylglycerol (PG) and phosphatidylinositol (PI) remained enigmatic for decades. PG and PI are uniquely secreted by alveolar epithelial cells, and their concentrations in this extracellular compartment greatly exceed that of any other tissue. Unexpectedly, studies of the innate immune Toll‐like receptors (TLRs) have revealed that PG and PI are potent antagonists of the activation of multiple TLRs including TLRs 1,2,3,4 and 6. The lipids bind to the receptors and prevent their activation by cognate ligands, and consequently prevent downstream activation of inflammatory signaling pathways, and release of inflammatory mediators such as interleukins 6 and 8, interferon‐lambda, eicosanoids and TNF‐alpha. In addition to suppressing activation of TLRs, the lipids also inhibit infection by major respiratory viruses including respiratory syncytial virus (RSV) and influenza A virus (IAV). Both PG and PI prevent the viral infections in cultured cells, and in vivo in mice. PG also completely abrogates lethal challenge of mice with pandemic H1N1‐IAV. Additional studies with ferrets further demonstrate protection against pandemic H1N1‐IAV. The lipids act as decoy receptors for both RSV and IAV, and block virus attachment to epithelial cell surfaces. We have constructed a library of 25 analogs of POPG and PI, which vary in both the structure of the polar headgroup and their hydrophobic moieties. Some of these analogs exhibit improved pharmacological properties compared with PG and PI. These studies demonstrate regulation of innate immune processes by pulmonary surfactant phospholipids, and also reveal their potential use for suppressing inflammatory processes in the lung and inhibiting establishment and spread of respiratory viral infections.

Support or Funding Information

Supported by NIH Grants HL 094629, U19‐AI 125357, GM 118819

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
3D Neuroanatomy: Using the HoloLens for an augmented reality approach in neuroanatomy education
Authors: Holman Parker , Bodnar Tamara , Ghomi Mehrdad , Choi Robyn , Moukhles Hakima , Krebs Claudia
Published: June 2021

The gold standard for neuroanatomy instruction involves the use of human brain specimens and prosections; however, there is still a clear need for innovative visualizations and educational tools that supplement dissection and improve learning outcomes for students in many areas including health related disciplines. With technological advances in computer imaging and display devices, anatomy educators increasingly have a variety of options for delivering educational content. There are limitations to these approaches, which include challenges in accurately representing 3D structures, ease of use for instructors and students, as well as oversimplification or inaccurate information. Students consistently struggle with integrating 2D images into a complex representation of the interconnected brain, and overlaying this understanding with important vascular and clinically relevant information. Increasingly, 3D technologies are being employed to help close this gap in student learning; however, not all 3D technologies are alike, nor do they offer the same instructional value.

The goal of the current project was to develop an augmented reality (AR) teaching tool that could be implemented in neuroanatomy instruction, and to evaluate its effectiveness in the classroom. In collaboration with Microsoft (BigPark Vancouver and Microsoft Garage Interns), an interactive lecture was developed using the HoloLens, a mixed reality headset offering the unique ability to blend virtual 3D content with the real world. Specifically, 3D reconstructions of basal ganglia nuclei were obtained from MRI scans and built into the teaching tool deployed on the HoloLens. Taking advantage of the HoloLens gesture recognition input system, interactivity was optimized, allowing the 3D reconstructions to be manipulated with ease and built up separately, as well as to visualize 2D MRI scans juxtaposed against 3D volumes of key brain nuclei. We are evaluating the pedagogical efficacy of this HoloLens teaching tool for undergraduate students, compared to traditional instructional methods. Assessments comprised multiple‐choice questions and structure identification on brain specimens focused on spatial orientation within the CNS as well as the ability to orient a 2D slice to the 3D structure of the brain. Students in both groups received baseline testing to assess both previous content knowledge as well as visuospatial reasoning to control for individual differences.

Support or Funding Information

PJH, TSB supported by Mitacs Accelerate Internships in partnership with Microsoft. Equipment donated from Microsoft. MG, RC supported by Emerging Media Lab, UBC Studios.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RNA and RNP Structures that Contribute to Viral Pathogenesis
Authors: Tolbert Blanton S.
Published: June 2021

RNA viruses persist to pose serious threats to human health and global economies. Disease progression mediated by viral pathogenesis requires numerous intersections between host proteins and viral RNA (vRNA) elements. Host‐vRNA complexes drive essential processes in the replication cycles of viruses; as such, they represent novel targets for therapeutic intervention. Members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family are cellular proteins often usurped by RNA viruses. My lab studies the molecular mechanisms by which HIV and Enterovirus 71 redirects hnRNPs to control viral gene expression. To that end, we have designed an integrated biophysical framework to elucidate the structures of vRNAs and protein‐vRNA complexes. Here, we describe 3D structures of conserved HIV and EV71 RNA elements that regulate splicing and translation. We further show that conformational adaptation and slow protein motions are fundamental properties by which hnRNP A1 and hnRNP H achieve cognate RNA recognition. To globally identify determinants of specificity, we used High Throughput Sequencing Equilibrium (HTS‐EQ) binding experiments to characterize the complete affinity distribution of human hnRNP A1 bound to the HIV ESS3 stem loop. HTS‐EQ involves competitive binding of hnRNP A1 to a completely randomized pool of ESS3 apical loop variants (n=16,384). Quantitative analysis of the resulting affinity distribution reveals RNA sequence; motif copy number; motif spacing; and secondary structure determine specificity by modulating the rates of productive hnRNP A1‐RNA encounters. Collectively, our work is offering mechanistic insights into the strategies by which viruses coopt cellular RNA binding proteins.

Support or Funding Information

NIH R01 GM101979

NIH U54 GM103297

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
From Petabytes to Molecular Insights: Building an Operating System for Cancer
Authors: White Kevin
Published: June 2021

Over the last decade there has been a confluence of technological advances in the computational and biological sciences that set the stage for large‐scale improvements in how cancers are diagnosed and treated, leading to a new vision for advancing precision oncology. As part of this vision we have taken steps to develop technological solutions and novel approaches to the challenge of integrating massive datasets and validating discoveries. In this context I will describe a series of proof‐of‐principle projects that our team has made to highlight basic discoveries and applications in cancer diagnostics and therapeutics. With these and many other efforts as a backdrop for what can be done, I will also describe our recent efforts at Tempus Labs to engineer and industrialize a systematic solution that is allowing physicians to treat patients enabled by a real‐time learning system that condenses petabytes of data into actionable information matched uniquely to each patient.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Suppression of Chemotherapy‐induced Cytokine/Eicosanoid Storm and Ovarian Tumor Growth by a Dual COX‐2/sEH Inhibitor
Authors: Gartung Allison , Yang Jun , Fernandes Djanira , Chang Jaimie , Hwang Sung Hee , Huang Sui , Kieran Mark , Hammock Bruce , Panigrahy Dipak
Published: June 2021

While chemotherapy remains a mainstay in cancer treatment, growing evidence indicates that it may stimulate tumor growth. Other cancer therapies including radiation, immunotherapy, and stem cell transplantation may also trigger the release of pro‐inflammatory and pro‐tumorigenic cytokines in the tumor stroma. We recently demonstrated that although cancer therapy reduces tumor burden by killing tumor cells, the resulting dead cells, or ‘debris', can promote tumor growth by stimulating the release of cytokines. Chemotherapy fails to generate remission in over 70% of patients with ovarian cancer, is rarely curative, and simultaneously creates tumor cell debris. Thus, cancer therapy is inherently a double‐edged sword and targeting a single cytokine or pathway may not prevent therapy‐induced cancer. Soluble epoxide hydrolase (sEH) inhibitors have been shown to stimulate the resolution of inflammation by promoting the synthesis of pro‐resolving mediators and counter‐regulating pro‐inflammatory cytokines. In this study, tumor cell debris was prepared in vitro by treating either murine or human ovarian tumor cells with first‐line platinum or taxane‐based cytotoxic chemotherapies used for treating ovarian cancer (e.g. cisplatin, carboplatin, or paclitaxel). Chemotherapy‐generated tumor cell debris stimulated ovarian tumor growth in both immunocompetent and immunocompromised hosts. Debris triggered macrophage production of a series of pro‐inflammatory and pro‐angiogenic cytokines. Further, LC‐MS‐MS–based oxylipin profiling of tumor cell debris generated by chemotherapy revealed a pathological release of tumor‐promoting bioactive lipids (‘eicosanoid storm’) including cyclooxygenase (COX)‐derived prostaglandins, lipoxygenase‐derived HETEs, CYP450‐derived DiHOMEs and EpOMEs. We hypothesize that dual COX‐2/sEH inhibition may be a novel modality in suppressing ovarian tumor growth by stimulating the natural debris‐clearing process. A dual COX‐2/sEH inhibitor, PTUPB, suppressed debris‐induced cytokines (e.g. TNFα, CCL2, CCL4, CXCL2, G‐CSF, ICAM‐1, MMP‐9, and PAI‐1) and eicosanoids (e.g. PGF2a, PGD2, and PGJ2). PTUPB also delayed the onset of debris‐stimulated tumor growth in an ovarian cancer (ID8) model, achieving sustained survival over 80 days post‐injection. It is imperative to overcome the predicament between killing tumor cells and the inherent tumor‐promoting activity of the debris. Thus, dual inhibition of COX‐2/sEH may be a novel approach in cancer therapy to suppress the therapy‐induced cytokine/eicosanoid storm and debris‐stimulated tumor growth.

Support or Funding Information

National Cancer Institute grant RO1 01CA170549‐02; ROCA148633‐01A4; NIEHS Superfund Research Program RO1

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hypoxanthine as a Checkpoint Metabolite in Epithelial Energy Modulation and Barrier Function
Authors: Lee J Scott , Wang Ruth , Alexeev Erica , Lanis Jordi , Colgan Sean
Published: June 2021

A primary function of intestinal epithelial cells (IECs) is to provide a selectively permeable barrier to protect the host from luminal microbiota and antigens, and to mediate the flux of nutrients, fluid, and waste across that barrier. Loss of intestinal barrier function is a hallmark of inflammatory bowel disease (IBD). Dysregulation of IEC barrier in IBD coincides with profound shifts in metabolic energy, especially in the colon, which exists in an energetically‐vulnerable state of physiologic hypoxia. The precise mechanism(s) by which metabolic pathways contribute to and control inflammation have yet to be elucidated. To delineate barrier‐related energy flux, an HPLC‐based profiling method was developed to track changes in energy and adenylate metabolites during intestinal epithelial barrier development and restitution. Cultured epithelia demonstrated a remarkable capacity to maintain ATP and phosphocreatine during barrier development. Further epithelial cell profiling identified a significant capacity for cultured human IECs to salvage hypoxanthine (Hpx) and shift their metabolic equilibrium toward the production of ATP and phosphocreatine. In a consistent manner, we have demonstrated that Hpx‐mediated shifts in metabolism result in enhanced epithelial barrier formation, increased epithelial restitution, and improved wound healing. Modulation of cellular energetics by Hpx were recapitulated with in vivo murine models, where orally‐delivered Hpx increased colonic ATP levels. Guided by an unbiased microarray analysis of Hpx treated human IECs, we identified a number of target genes important in mucosal differentiation, energy metabolism, and reactive oxygen species mediation. As the ATP consumed by IECs under energetic stress is shuttled to and pooled as Hpx, it appears not only as a central energy metabolite, but also as a regulator of gene expression. Taken together, our ongoing studies provide strong evidence that Hpx functions as a central, checkpoint energy metabolite in IEC function.

Support or Funding Information

NIH/NIDDK

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Desmoglein Binding Properties are Regulated by Plakophilins
Authors: Vielmuth Franziska , Fuchs Michael , Foresti Marco , Keil Rene , Hatzfeld Mechthild , Spindler Volker , Waschke Jens
Published: June 2021

Desmosomes are highly organized protein complexes required for strong intercellular adhesion of keratinocytes. To maintain desmosome integrity, desmosomal cadherins provide intercellular mechanical coupling with their extracellular domains whereas plaque proteins such as plakophilins (Pkp), plakoglobin and desmoplakin mediate the anchorage to the intermediate filament cytoskeleton. In addition, Pkp isoforms are involved in the regulation of desmosomal turnover and several signaling pathways and are thereby crucial for desmosomal adhesion. However, their contribution to binding properties of desmosomal cadherins has not been elucidated yet. Here, we used murine keratinocytes lacking either Pkp1 or Pkp3 (Pkp1 k.o. / Pkp3 k.o.) and characterized Dsg1 and 3 binding properties in comparison to wildtype cells (Pkp wt). Both, Pkp1 and 3 k.o. cells showed compromised intercellular adhesion and reduced membrane levels of Dsg1 and Dsg3. Consistent with these findings, the amount of Dsg3 oligomers detected by membrane‐impermeable extracellular crosslinking was drastically reduced in both, Pkp1 and 3 k.o. cell lines. Along the same line, binding frequencies of both, Dsg1 and Dsg3‐dependent binding events as well as binding strength and the step position of Dsg3 interaction, the latter of which represents a measure for cytoskeletal anchorage, were drastically reduced in Pkp1 and 3 k.o. cell lines. Further, Dsg1 binding frequency was higher at cell borders compared to cell surface in wt but not in Pkp1 and Pkp3 k.o. cells, indicating a Pkp‐dependent control of Dsg1 membrane positioning. Moreover, alteration of p38MAPK signaling, a central signaling pathway for the regulation of desmosomal adhesion, correlated with intercellular adhesion in all cell lines. However, Pkp3 k.o. cells showed reduced activity of p38MAPK under untreated conditions as well as a diminished response to the p38MAPK activator anisomycin, indicating an important role of Pkp3 for p38MAPK signaling. Taken together, these data suggest (1) that Pkps are important for localization and binding of desmosomal cadherins and (2) that Pkp3 may participate in the regulation of desmosomal adhesion via p38MAPK signaling.

Support or Funding Information

Funding Information: Supported by Else‐Kröner‐Fresenius‐Stiftung (2016_A157).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deficiency of miR‐1954 promotes cardiac remodeling
Authors: Gupta Sudhiranjan , Chisson Valorie
Published: June 2021

Cardiac remodeling due to hemodynamic overload is associated with significant morbidity and mortality. In response to stress, cardiomyocyte (CM) become hypertrophied whereas cardiac fibroblasts convert into myofibroblasts. The phenomenon leads to the development of cardiac hypertrophy, fibrosis and impair cardiac function. Previously, we have shown the pivotal role of miRNA (a new class of post‐transcriptional regulators) in cardiac remodeling, but, loss of miRNA contributing to the onset of cardiac remodeling remains elusive. Using next generation miRNA sequencing, we discovered a panel of novel dysregulated miRNAs from read‐data, secondary structure and miRPara classification score analysis in wild‐type mice (WT) infused with Angiotensin II (Ang II). Among them, one was identified as miR‐1954, a novel miRNA which was significantly reduced in Ang II‐infusion and transverse aortic constriction (TAC). Following an unbiased approach, we confirmed that Sp1‐Gata4‐Col I‐Tsp1‐axis is the bona‐fide targets. Our hypothesis is that deficiency of miR‐1954 exacerbates cardiac remodeling leading to hypertrophy and fibrosis through paracrine mechanism; and overexpression of miR‐1954 mitigates the cardiac damage and abrogates remodeling by modulating Sp1‐Gata4‐Col I‐Tsp1‐axis. Our data demonstrated that depletion of miR‐1954 in CM triggers hypertrophic response by modulating Sp1 and Gata4; releases soluble factors (Tgfb1) that triggers cardiac fibroblasts proliferation; upregulation of thrombospondin 1 (Tsp1) and collagen I (Col I). Overexpression of miR‐1954 in CM reverses these processes implicated a cellular cross‐talk. Cardiac‐specific overexpression of pre‐miR‐1954 transgenic mice (miR‐1954 Tg) showed reduced cardiac mass and improved function compared to WT littermate after Ang II treatment. Inhibition of miR‐1954 by locked nucleic acid of anti‐miR‐1954 exacerbates cardiac hypertrophy and fibrosis. Our findings provide evidence that loss of miR‐1954 promotes cardiac remodeling by targeting Sp1‐Gata4‐Col I‐Tsp1‐axis and, overexpression of miR‐1954 reverses the process. We conclude that miR‐1954 could be a triggering factor in cardiac remodeling and providing new mechanistic information for therapeutic benefit.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intersectional Genetic Approaches for Studying Dopaminergic Subpopulations
Authors: Bateup Helen
Published: June 2021

Midbrain dopamine neurons project to numerous targets throughout the brain to modulate various behaviors and brain states. Within this small population of neurons exists significant heterogeneity based on circuitry, physiology, and disease susceptibility. Recent studies have shown that dopamine neurons can be subdivided based on gene expression; however, the extent to which genetic markers define functionally relevant dopaminergic sub‐types has not been determined. To address this, we performed single cell RNA‐sequencing of midbrain dopamine neurons and identified genes that define dopaminergic subpopulations. Using a combination of anatomy, circuit tracing, and electrophysiology we have defined the projection targets, cellular properties, and disease vulnerability of two groups of dopamine neurons. Our current efforts are aimed at generating intersectional mouse genetic tools that will allow us to selectively target and manipulate dopaminergic subpopulations to understand their functional roles. This approach will allow us to dissect the complexity of the dopaminergic system, thereby furthering our understanding of how specific dopaminergic circuits control behavior in both normal and disease states.

Support or Funding Information

This work is supported by a NARSAD Young Investigator Grant from the Brain & Behavior Research Foundation and a fellowship from the Alfred P. Sloan Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cerebellar Neuron Diversity in the Zebrafish: More atoh1 Genes for More atoh1 Fates?
Authors: Moens Cecilia B. , Kidwell Chelsea U.
Published: June 2021

During mammalian cerebellar development, a single Atoh1 basic‐helix‐loop‐helix transcription factor is required sequentially for the specification of multiple neuron types. The zebrafish genome encodes three paralagous atoh1 genes, atoh1a, atoh1b and atoh1c with overlapping but distinct spatio‐temporal expression patterns in the developing cerebellum. (The presence of extra paralogs in fish genomes is due to a whole‐genome duplication at the base of the teleost lineage). We have studied the roles of zebrafish atoh1 paralogs using transgenic reporters to mark atoh1c‐and atoh1a‐specified neurons, as well using CRISPR/Cas9‐generated mutant alleles in all three genes. Our analysis suggests that additional atoh1 genes are the source of additional neuronal diversity in the zebrafish cerebellum. atoh1c‐expressing cells are organized in two distinct domains: an early expression domain at the mid‐hindbrain boundary gives rise to an extracerebellar population in rhombomere 1 and an upper rhombic lip domain gives rise to granule cells that populate the four granule neuron territories of the fish cerebellum. Atoh1a‐expressing cells at the URL give rise to a separate population of granule neurons that do not overlap with the atoh1c populations. Consistent with this, atoh1a and atoh1c are both required for the specification of the full complement of granule neurons. These results suggest that zebrafish atoh1 duplicates generate granule neuron diversity that is not detected in mammals.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reconstructing Jaw Movement and Musculature in the Xenacanth Shark Orthacanthus texensis
Authors: Bronson Allison Whitney , Heers Ashley
Published: June 2021

Most of the shark fossil record consists of teeth, due to their relative abundance, decay‐resistant enameloid, and the poor preservation potential of cartilaginous skeletons. However, rare cartilage fossils have changed our understanding of chondrichthyan evolution. Sharks have traditionally been considered primitive vertebrates, but new paleontological information demonstrates that chondrichthyans are actually a highly specialized group of fishes. In addition to valuable phylogenetic characters from fossil cartilage, three‐dimensionally preserved specimens allow us to explore functional morphology in extinct taxa. Here, we reconstruct jaw movement and musculature of the xenacanth Orthacanthus texensis. The cranium and jaws of Orthacanthus have been previously described, but a complete three‐dimensional specimen from the Permian of Texas (MCZ 12872) has never been addressed in its entirety. To better understand xenacanth anatomy, we took three‐dimensional surface scans of Orthacanthus skeletal elements by laser scanning, and articulated each element in Maya. We reconstructed musculature based on comparisons with several dissections of extant taxa, and then used Software for Interactive Musculoskeletal Modeling (SIMM) and OpenSim to assess muscle moment arms, potential functions, and optimal positioning for increasing bite forces. We find that Orthacanthus had large, extensive adductor muscles. To our knowledge, this is the first time SIMM has been used to model extinct chondrichthyan musculature. Computer modeling is an important method for this type of study, as xenacanths are not closely related to any modern sharks, so dissection of extant taxa alone is inadequate for modeling xenacanth musculature.

Support or Funding Information

Richard Gilder Graduate School at the American Museum of Natural History

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Model to Assess the Effects of Osteoporosis Medications on Bone Quality
Authors: Sumner D. Rick , Ross Ryan
Published: June 2021

In order to examine the effects of osteoporosis treatments on bone tissue that forms during drug administration, we have adapted a decades old model in the rat(1, 2). The premise of our work is that bone matrix mineralization within cortical bone is an important but oft‐neglected component of bone remodeling. Aberrant mineralization can reduce bone strength and increase fracture risk independent of changes in bone mass. Sodium fluoride represents an example of an osteoporosis treatment that alters the mineralization process, leading to increased fracture risk in spite of increased bone mass, thereby causing the very thing it was meant to cure. Unfortunately, small animal models to study matrix mineralization during remodeling of cortical bone in the adult skeleton did not exist when this drug was under development; so these unexpected effects were only discovered in patients.

We have performed a series of studies to develop and validate a rodent model to study matrix mineralization during remodeling in adult cortical bone. Briefly, remodeling is induced by severely restricting dietary calcium in lactating animals (0.01% as opposed to the normal 0.6%). When the pups are weaned, the dams are immediately returned to the normal Ca diet. We study the bone that forms during the post‐weaning period in the dams. We have performed experiments to assess the most appropriate anatomical site for examining intra‐cortical remodeling and the effects of manipulating dietary Ca on mineral metabolism and mineralization kinetics. We found that the border between the distal femoral diaphysis and metaphysis is the best site for sampling intra‐cortical remodeling because this site had the most induced pores of a size consistent with rat secondary osteons(3). The increase in porosity was resolved 30 days after weaning, suggesting that the induced intra‐cortical resorption spaces had totally infilled. Endocortical remodeling can also be examined at this site. Studies using in vivo fluorochrome labeling, Fourier‐transform infrared microspectroscopy, back‐scatter scanning electron microscopy, microcomputed tomography, histology and serum assays indicate that matrix mineralization occurs normally and largely within a normal endocrine environment despite the manipulation of Ca in the diet. A major advantage of this model is that it provides multiple sites of synchronized matrix mineralization. We are currently using the model to study how common and emerging osteoporosis drugs affect matrix mineralization and mechanical behavior.

Support or Funding Information

NIH Grant R21AR065604

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pathophysiology of Anti‐resorptive Medication‐related Osteonecrosis
Authors: Aghaloo Tara
Published: June 2021

Osteonecrosis of the jaws (ONJ) is a devastating complication of potent antiresorptive medications utilized for benign and malignant disorders of bone metabolism. Bisphosphonate related ONJ (BRONJ) was first identified in 2003 and 2004, and its prevalence is reported between 0.8–12%. Although significant progress has been made in our understanding of the disease, much more work needs to be done to completely explain its pathophysiology. Many hypotheses have been proposed, which have sparked empirically‐based treatment modalities. It is most likely that the pathophysiology of ONJ is multifactorial, where inflammation/infection and inhibition of bone remodeling are necessary for development of the disease. This presentation will present current evidence for ONJ pathophysiology, and prevention and treatment approaches that can be implemented in the dental office.

Support or Funding Information

NIH/NIDCR

Amgen

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
When PNS Assembly Goes Awry: Familial Dysautonomia
Authors: Lefcort Frances , Chaverra Marta , George Lynn
Published: June 2021

Over a century of elegant work has contributed to our understanding of how neural crest cells migrate throughout the embryo to give rise to the majority of cell types in the peripheral nervous system (PNS). This rich foundation is the basis for our lab's investigation of the cellular and molecular mechanisms that go awry to result in the developmental disorder of the PNS, Familial dysautonomia (FD). Three of the key clinical hallmarks of FD include a reduction in pain and temperature sensation, misregulation of the sympathetic nervous system, and impaired gastrointestinal tract function. The neurons in all of these systems derive from the neural crest. Consequently, a prevailing hypothesis in the field was that FD resulted from faulty neural crest migration. To test this hypothesis, we generated mice in which the gene that is mutated to cause FD, IKBKAP/ELP1, was deleted selectively from the neural crest. Interestingly, by the end of embryogenesis, we found that in fact the number of pain and temperature receptors in the dorsal root ganglia were significantly reduced as were sympathetic neurons in the superior cervical ganglia, recapitulating the human disease phenotype (George et al., 2013, PNAS). However, these studies also demonstrated that this selective reduction in neuronal number was not due to faulty neural crest cell migration. Rather, the reductions were due to (1) the death and premature differentiation of the Pax3+ pain and temperature progenitor cells and (2) exacerbated apoptosis of TrkA+ pain and temperature receptors. Interestingly, the TrkC+ proprioceptors developed normally, although they later die progressively in adulthood (Elisabetta Morini and Susan Slaughenhaupt, personal communication). Upon the completion of neurogenesis in the dorsal root and sympathetic ganglia, PNS axons must innervate their targets to fully differentiate. Our work and others (Jackson et al., 2014, Development; Abashidze et al., 2015, PLoS One) demonstrate that IkbkapElp1 is required for normal axon outgrowth and target innervation. These and other findings for how and why PNS development is disrupted in FD will be discussed.

Support or Funding Information

NIH R01 NS086796; Dysautonomia Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Authentic Anatomy Education: Leave your Ego at the Door to Create Memorable Learning Experiences
Authors: O'Loughlin Valerie Dean
Published: June 2021

As teachers, we strive to inspire interest and wonder in the field of anatomy for our students. We seek to clarify the complex, we try to scaffold learning, and in so doing we facilitate the development of well‐grounded anatomical knowledge in our students. We also serve as mentors to our graduate students who are progressing along the path of future anatomy educators. Yet as we acquire our knowledge and experience, paradoxically we may become less relatable to our students, as they have not seen our hard fought path, mired with mistakes, to our current destination. In order to create authentic anatomy learning experiences and inspire a growth mindset in our students about learning, instructors should mention their past mistakes and failures, so as to let students know we have shared their struggles and have persisted. Likewise, as we train future anatomy educators, we must make visible our path from novice instructor to experienced educator, so as to instill a growth mindset in our future colleagues. Making these paths visible sometimes requires ‘leaving your ego by the door’ in order to create memorable learning moments. Here, I share some of my own examples of creating authentic anatomy learning experiences for my students and future anatomy educators.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Single protein‐catalyzed ubiquitination: Chemistry and implications in cell signaling
Authors: Luo Zhao‐Qing
Published: June 2021

Posttranslational modification by the 76‐residue ubiquitin molecule participates in virtually every cellular process in eukaryotic cells, whereby it regulates the activity of substrate proteins by altering their turnover time, cellular localization or biochemical function. Classical ubiquitination is catalyzed by the action of E1, E2 and E3 enzymes that function coordinately to activate, conjugate and ligate the modifier to a protein substrate. We recently found that members of the SidE effector family such as SdeA catalyze ubiquitination a mechanism that completely differs from the canonical three‐enzyme cascade. In this scenario, ubiquitin is activated by ADP‐ribosylation at Arg42 (ADPR‐Ub) via a mono‐ADP‐ribosyltransferase (mART) motif. Subsequent works by others showed that the reaction intermediate is then utilized by a phosphodiesterase (PDE) activity also embedded in SdeA, which cleaves ADPR‐Ub to transfer the phosphoribosylated ubiquitin moiety to serine residues of the substrate. In this talk, I will discuss our recent findings on the functional significance of SidEs‐induced ubiquitination on host proteins. I will also discuss a new mechanism of ubiquitination catalyzed by another L. pneumophila effector and its implications in cell signaling.

Support or Funding Information

NIH‐NIAID R01127465

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Conformational Regulation of Phospholipase C Enzymes
Authors: Lyon Angeline , Garland‐Kuntz Elisabeth E. , Vago Frank S. , Van Camp Michelle , Sieng Monita , Corpstein Clairissa , Blaine Arryn T. , Jiang Wen
Published: June 2021

Phospholipase C (PLC) enzymes, in particular the PLCβ and PLCɛ subfamilies, are essential for normal cellular function in the cardiovascular system where they generate second messengers in response to extracellular signaling that increase calcium levels and activate protein kinase C. Crystal structures of PLCβ have provided some insights into its architecture and regulation, but there is no published structural data for PLCɛ or any catalytically active fragment of the enzyme. We have used small angle X‐ray scattering, electron microscopy, and biochemical assays to begin characterizing the solution structures and regulation of PLCɛ and PLCβ. We find that PLCɛ is conformationally dynamic in solution and that its PH domain is essential for maximum basal activity. We also find PLCβ adopts multiple conformations in solution, wherein its PH domain and EF hands are extended away from the catalytic core. Using site‐directed mutagenesis and functional assays, we find that stabilization of the PH domain in its extended or closed conformation impacts its activity. Taken together, our data support a more complex and global model for PLC regulation, wherein the conformation of the PH domain and EF hands relative to the catalytic core modulates its lipase activity at the membrane.

Support or Funding Information

This work is supported by an American Cancer Society Institutional Research Grant (IRG‐14‐190‐56) to the Purdue University Center for Cancer Research and an American Heart Association Scientist Development Grant 16SDG29930017 to A.M.L.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MECHANISMS OF THE IDH1/2 MUTATIONS AND ITS ASSOCIATION WITH CONTRADICTORY SURVIVAL OF GLIOBLASTOMA PATIENTS VERSUS AML PATIENTS
Authors: van Noorden Cornelis J.F. , Khurshed Mohammed , Hira Vashendriya V.V. , Molenaar Remco J.
Published: June 2021

Isocitrate dehydrogenase 1 and 2 (IDH1/2) is mutated (IDH1/2mt) in various types of human cancer such as glioma and acute myeloid leukemia (AML), a structural alteration that leads to catalysis of α‐ketoglutarate to the oncometabolite D‐2‐hydroxyglutarate (D2HG). Heterozygous IDH1/2mt cancer cells produce less IDH1/2‐mediated NADPH, such that after exposure to ionizing radiation (IR) there were higher levels of reactive oxygen species, DNA double‐strand breaks and cell death compared to IDH1 wild‐type (IDH1wt) cells. These effects were reversed by the IDH1mt inhibitor AGI‐5198. Exposure of IDH1wt cells to D2HG was sufficient to reduce IDH‐mediated NADPH production and increase IR sensitivity. Mechanistic investigations revealed that the radiosensitivity of heterozygous cells was independent of the well‐described DNA hypermethylation phenotype in IDH1mt cancers. Thus, our results argue that altered oxidative stress responses are a plausible mechanism to understand the radiosensitivity of IDH1mt glioma. Further, they offer an explanation for the relatively longer survival of patients with IDH1mt glioma, and they imply that administration of IDH1mt inhibitors in these patients may limit irradiation efficacy in this setting.

Whether this also holds true for IDH1/2mt AML was investigated using primary human IDH1mt, IDH2mt and IDH1/2wt AML cells. It was investigated whether IDH1/2 mutations cause increased DNA damage and sensitization to daunorubicin, irradiation, and the PARP inhibitors olaparib and talazoparib. IDH1/2mt inhibitors protected against these treatments. Combined treatment with a PARP inhibitor and daunorubicin had an additive effect on killing of IDH1/2mt AML cells. We provide evidence that the therapy sensitivity of IDH1/2mt cells was caused by D2HG‐mediated downregulation of expression of the DNA damage response gene ATM and not by altered redox responses due to metabolic alterations in IDH1/2mt AML cells. Thus, IDH1/2mt AML cells are sensitive to PARP inhibitors as monotherapy but especially when combined with a DNA‐damaging agent such as daunorubicin, whereas concomitant administration of IDH1/2‐mutant inhibitors during cytotoxic therapy decreases the efficacy of both agents in IDH1/2mt AML cells.

Support or Funding Information

This study was financially supported by the Dutch Cancer Society (KWF; UVA 2014‐6839; CJFVN, MK, VVVH, RJM)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The influence of high fiber diet and probiotic supplementation in a murine model of colitis
Authors: Owens Josh , Saeedi Bejan , Darby Trevor , Jones Rheinallt
Published: June 2021

Probiotics are therapeutic agents administered to patients with IBD. In addition, it has recently been hypothesized that a high fiber diet may synergize with probiotics to exert an elevated cytoprotective effect on host tissue during colitis. Thus, we investigated the extent to which both orally administered probiotics and a high fiber diet protected intestinal tissue in a murine model of colitis. We administered the well‐studied probiotic, Lactobacillus rhamnosus GG (LGG) to 8 week‐old mice for two weeks before a 7‐day challenge with 3% Dextran Sodium Sulfate (DSS). Protection was assessed in terms of disease activity index and histology. Mice given LGG or a high fiber diet conferred modest protection from DSS‐induced colitis compared to controls. However, the cytoprotective effect was significantly strengthened in mice fed a high fiber diet in conjunction with LGG. Flow Cytometry analysis of immune cells in the colonic lamina propria revealed an immune‐modulatory effect of LGG, which was elevated under a high fiber diet. Together, these data suggest that a high fiber diet plus a probiotic can lead to enhanced cytoprotection against DSS‐induced colitis compared to either LGG feeding, or a high fiber diet alone.

Support or Funding Information

Research supported by NIH grant R01DK98391

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.