bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of Altered Metabolic States in Cancer and Other Human Diseases
Authors: DeBerardinis Ralph J.
Published: June 2021

Altered metabolism is a rich source of phenotypic diversity throughout nature, and it contributes to a very large number of human disease phenotypes, including cancer and pediatric inborn errors of metabolism (IEMs). In cancer, mutations in oncogenes and tumor suppressors reprogram metabolic preferences in malignant cells so as to enhance their ability to grow and withstand stress within tumors. IEMs constitute the single largest category of human genetic diseases. They are caused by germline defects in genes encoding metabolic enzymes and nutrient transporters, and they result in chronic, sometimes fatal dysfunction of the brain, liver and other organs, usually manifesting first in children. Crucially, in both cancer and IEMs, understanding the underlying metabolic perturbation and its relationship to the phenotype can produce opportunities for therapeutic intervention. Thus, applying modern, comprehensive techniques in metabolic analysis to human subjects could translate into better clinical management of a number of diseases. I will discuss the use of metabolomics (profiling of steady‐state metabolite abundances) and metabolic flux analysis (the use of stable‐isotope tracers to measure flow through metabolic pathways in living tissue) in human subjects with cancer and IEMs to understand the metabolic basis of tissue dysfunction, to identify therapeutic opportunities, and to discover entirely new human disease phenotypes.

Support or Funding Information

Howard Hughes Medical Institute (Faculty Scholars Program), National Cancer Institute, Robert L. Moody Faculty Scholar

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Virion display (VirD) approach to characterizing non‐odorant GPCRs in Humans
Authors: Zhu Heng , Desai Prashant , Syu Guan‐Da
Published: June 2021

Approximately one‐third of the human proteome is comprised of membrane proteins that belong to protein families with a wide variety of biochemical activities, such as transporters, channels, receptors, recognition molecules, and adhesion molecules. Among them, the GPCR protein family is of particular importance because they exhibit a wide variety of signaling pathways depending on ligand binding specificity and various coupling proteins during activation. In deed, they are most preferred drug targets. However, GPCRs are notoriously difficult to study because they have to be embedded in a membrane to maintain a native conformation and many require proper posttranslational modifications (PTMs), such as glycosylation. To facilitate high‐throughput biochemical analyses of GPCRs, we have recently developed a VirD array technology by which a GPCR is displayed in the membrane envelop of herpes simplex (i.e., HSV‐1) virions to maintain their native conformation. To date, we have displayed 315 non‐odorant GPCRs on the virions and formed a GPCR VirD array by spotting down the purified viruses. To characterize the virion‐displayed GPCRs, we first tested the orientation and ligand binding activities of selected GPCRs. Of the 20 commercial mAbs targeting ectodomains of GPCRs, 11 could specifically recognize their intended targets on the VirD arrays. For the nine GPCRs that failed to be recognized by the commercial mAbs, we showed that they could readily recognize their canonical ligands using single molecule imaging. Moreover, using an SPR‐based technology, we could obtain real‐time binding kinetics and deduce binding affinity for selected ligand‐GPCR pairs, indicating that they were all folded correctly. To determine whether VirD technology could help de‐orphanize GPCRs, ~80 fluorescently labeled orphan GPCR virions were individually probed to HuProt array, each comprised of 20,240 human proteins. Many secreted and transmembrane proteins were identified as specific targets, providing an important stepping‐stone for further characterization of these orphan GPCRs. Taken together, the VirD technology provides a high‐throughout platform for screening for drugs, affinity reagents, ligands, and beyond.

Support or Funding Information

NCI IMAT R33 and CDI Labs

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression of Sirpα Tailless Mutant in Mice Impairs Naïve CD4+ T Cell Adhesion to Immobilized ICAM‐1 and TCR Induced Proliferation.
Authors: Planas Francisco Esteban Velázquez , Autio Anu Esteban , Newton Gail , Parkos Charles , Luscinskas Francis William
Published: June 2021

Signal Regulatory Protein alpha (SIRPα) interaction with its ligand CD47 plays an important role in immune cell homeostasis. CD47 is expressed by essentially all cell types, and acts as a “don't eat me signal” and a marker of self. We previously demonstrated that CD47 on both endothelium and T cells plays an important role in T cell adhesion and diapedesis in vivo and in vitro, and that CD47−/− CD4+ T cells had impaired antigen mediated proliferation in a model of Experimental Autoimmune Encephalomyelitis (EAE). In contrast to CD47, SIRPα has been reported as selectively expressed in the Central Nervous System (CNS), in most myeloid cells and at very low levels‐to‐absent in lymphoid cells in humans. To gain insight into the function of SIRPα in leukocyte recruitment, Sirpα truncated cytoplasmic, non‐signaling mutant (Sirpatm1Nog) transgenic mice and WT mice were studied in TNFα‐induced dermal air pouch model of inflammation. Unexpectedly, CD4+ T cells in Sirpatm1Nog mice, compared to WT mice, had significantly decreased recruitment into the air pouch (WT, 11.9 ± 2.9 ×103 vs Sirpatm1Nog, 7.8 ± 2.6 ×103, p<0.05), whereas no differences in CD8+ T cell or myeloid cell recruitment were observed. Interestingly, flow cytometric analysis using P84 monoclonal antibody showed that a low but significant fraction of CD4+ T cells in both WT and Sirpatm1Nog mice expressed SIRPα. Expression of Very Late Antigen‐4 (VLA‐4) and Lymphocyte Function Associated Antigen‐1 (LFA‐1) integrins, which are ligands of Vascular Cell Adhesion Molecule‐1 (VCAM‐1) and Intercellular Adhesion Molecule‐1 (ICAM‐1), respectively, were similar in T cells from WT and Sirpatm1Nog mice. Additional studies showed naïve CD4+ isolated from Sirpatm1Nog, compared to WT mice, had significantly blunted proliferation 48 hrs after T cell receptor crosslinking (TCR‐XL), and that CD4 T cells had impaired SDF‐1α induced arrest on immobilized ICAM‐1 under shear flow conditions. In summary, these studies demonstrate that SIRPα plays an unexpected and important role in T cell recruitment in vivo and in vitro and in TCR‐XL induced proliferation in vitro. These data suggest that loss of SIRPα signaling in SIRPα+ T cells leads to impaired LFA‐1 activation.

Support or Funding Information

R01 HL125780 and T32HL007627.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Loss of local innervation induces lymph node expansion
Authors: Chen Chien‐Sin , Ince Louise , Scheiermann Christoph
Published: June 2021

Peripheral lymphoid and non‐lymphoid tissues receive direct innervation via the nervous system but the relevance of neural input for the mounting of immune responses remains largely unknown. Ablating local innervation to popliteal lymph nodes (popLNs) by unilateral surgical ligation of the sciatic nerve led to footpad edema and dramatic expansion of popLNs due to an increase in cellularity in the ipsilateral side. In contrast, local surgeries leaving popLNs innervated, such as femoral denervation or sciatic denervation downstream of the popliteal fossa, did not generate nodal expansion. The increase in lymph node cellularity could be attributed to B cell proliferation and was associated with enhanced lymphocyte homing from the blood while leukocyte egress into efferent lymph remained unchanged. Homing was a critical factor in this process since blocking leukocyte entry into the lymph node with antibodies directed against L‐selectin or both α4‐ and αL‐integrins ablated denervation‐induced nodal expansion. Higher levels of TNF‐α, IL‐17 and IL‐4 were detected in denervated lymph nodes, the latter of which was also observed by systemic or local sympathectomy. Thus, loss of neural input to lymph nodes resulted in increased cellularity which was dependent on leukocyte homing. These data provide evidence for an important role of direct neural innervation in the control of immune responses.

Support or Funding Information

Funded by the German Research Foundation (Emmy Noether SCHE 1645/2‐1 and SFB914 projects B09 and Z03) and the European Research Council (CIRCODE, 635872)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Surface glycocalyx and glypican‐1 mediate tumor cell metastasis
Authors: Qazi Henry , Moran Heriberto , Cancel Limary M. , Mayer Mariya , Roberge Sylvie , Huang Peigen , Munn Lance L. , Tarbell John M.
Published: June 2021

The surface proteoglycan/glycoprotein layer (glycocalyx) on tumor cells has been associated with cellular functions that can potentially enable invasion and metastasis. In addition, aggressive renal carcinoma cells (SN12L1) with high metastatic potential have enhanced invasion rates compared to low metastatic (SN12C) cells in response to interstitial flow stimuli in vitro. Our previous studies suggest that heparan sulfate (HS) and hyaluronic acid (HA) in the glycocalyx play an important role in this flow mediated mechanotransduction and upregulation of invasive and metastatic potential. In our recent study, SN12L1 cells were genetically modified to suppress HS production by knocking down its synthetic enzyme NDST1. Using modified Boyden chambers with defined interstitial flow, we showed that flow‐enhanced invasion is suppressed in HS deficient cells. We also examined two prominent HSPGs on renal carcinoma cells – glypican‐1 and syndecan‐1 and one prominent HA receptor – CD44. We observed higher glypican‐1 levels in flow dependent SN12L1 cells when compared to SN12C cells. Caki‐1 (highly metastatic) cells did not display flow‐dependent invasion in vitro and did not display elevated glypican‐1 compared to low metastatic Caki‐2 cells. However we did observe significantly increased HS, HA, syndecan‐1 and CD44 in Caki‐1 compared to Caki‐2 cells suggesting an alternative mechanism for reported higher metastatic rates in these cells. All of our data are consistent with the hypothesis that glypican‐1 is the core protein responsible for flow sensing in metastatic cancer cells. This is also consistent with observations in endothelial cells. To assess the ability of tumor cells to metastasize in vivo, parental or HS knockdown SN12L1 cells expressing fluorescent reporters were injected into kidney capsules in SCID mice. Histological analysis confirmed that there was a large reduction (95%) in metastasis to distant organs by tumors formed from knockdown cells compared to control cells with intact HS. The reduction was even greater (98%) in lungs where most of the metastases from the control cells were observed. The ability of these knockdown cells to invade surrounding tissue was also impaired. The substantial inhibition of metastasis and invasion upon reduction of HS suggests an active role for the tumor cell glycocalyx and glypican‐1 in tumor progression.

Support or Funding Information

Grant: RO1CA204949 Targeting glycocalyx mediated mechanisms of tumor metastsis

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dsg2 regulates intestinal epithelial cell adhesion and barrier function via EGFR
Authors: Ungewiß Hanna , Schlegel Nicolas , Waschke Jens
Published: June 2021

Rapidly renewing epithelial tissues such as the intestinal epithelium require precise modulation of intercellular adhesion to preserve barrier integrity during cell division and differentiation. Previously, we have shown that Desmoglein 2 (Dsg2), an adhesion molecule of desmosomes, beside its adhesion function, regulates intestinal barrier properties via p38MAPK signaling. Here we show that Dsg2 and epidermal growth factor receptor (EGFR) co‐localize at the apical part of cell‐cell contacts in polarized enterocytes using confocal and stimulated emission depletion (STED) microscopy. DLD1 cells deficient for Dsg2 display EGFR mislocalization and reduced protein levels of activated as well as total EGFR. Atomic force microscopy (AFM) revealed EGFR‐Dsg2 interaction both under cell‐free conditions as well as on living enterocytes, which was confirmed by co‐immunoprecipitation. Moreover, EGFR inhibition reduced cell cohesion and barrier reformation. In line with this, we demonstrate that Src activity is required for barrier formation and localization of EGFR and Dsg2 to cell‐cell contacts. Thus, our study reveals a new mechanism by which Dsg2 regulates barrier properties via EGFR.

Support or Funding Information

This work was funded by grants of the Deutsche Forschungsgemeinschaft: the priority program DFG_SPP1782

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antenatal exposure to secondhand smoke impacts growth and cardiopulmonary energetics in 4‐week‐old mice
Authors: Hirschi Kelsey M. , Egbert Kaleb , Clark Christian , Mella Nate , Plothow Edward , Mejia Juan F. , Arroyo Juan A. , Reynolds Paul R.
Published: June 2021

Secondhand smoke (SHS) is a well‐established cause of respiratory illness in infants and children who live in environments where exposure is common. Recent studies examining potential effects of antenatal SHS exposure suggest that antenatal exposure to SHS impacts cardiovascular and respiratory function through childhood and may have lifelong ramifications. In the present study, we sought to determine effects of antenatal SHS exposure on 4‐week‐old mice. C57/Bl6 mice (n=8) were exposed to SHS or room air via a nose‐only delivery system (Scireq) beginning on gestational day (dGA) 14.5 to 17.5 dGA. At 4 weeks of age, mice were weighted and blood pressure and heart rate was determined with a tail occlusion cuff (Kent Scientific). Whole body, heart/body and liver/body weights were determined. Lastly, organ‐specific mitochondrial function tests were performed. At 4 weeks of age, antenatal exposure of SHS caused: 1) a significant reduction (p<0.03) in total body weight; 2) significantly elevated systolic (p<0.0002) and diastolic (p<0.004) blood pressure; 3) no differences in the animal's heart rate; 4) significantly decreased heart (p<0.004) and kidney (p<0.0006) weights when indexed to body weight; and 5) significantly decreased oxygen consumption related to cellular respiration in SHS‐exposed hearts and lungs when compared to room air exposed controls. Our results indicate that antenatal exposure to SHS has a detrimental effect on growth rates and cardiopulmonary energetics through the 4th week of age, or the equivalent of early adolescence. These results may be beneficial in understanding the long‐term effects of antenatal SHS exposure.

Support or Funding Information

This work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI, PRR and JAA) and a BYU Mentoring Environment Grant (JAA and PRR).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NOCICEPTIVE STIMULUS IN THE NEONATAL PERIOD AFFECTS SATELLITE GLIAL CELLS MORPHOLOGY IN ADULT LIFE, WITH DIFFERENCES BETWEEN MALE AND FEMALE RATS
Authors: Simoes Ana Leda Bertoncini , Silva Greice Anne Rodrigues , Giorgetto Carolina , Carmo Elisabete Cassia , Fazan Valeria Paula Sassoli
Published: June 2021

Satellite glial cells (SGCs) are found in the dorsal root ganglia (DRG) surrounding completely and individually sensory neurons, forming a thin sheath cells with regulatory function of the neuronal microenvironment. During the nociceptive stimulus the SGCs are activated and express glial fibrillary acidic protein (GFAP). Sustained nociceptive stimulus may cause morphological changes in SGCs influencing the mechanisms of pain chronification that may differ between genders. Understanding the plasticity in peripheral pain pathways considering gender differences is crucial, particularly when noxious stimuli are applied to neonates, when the nervous system is still under rapid and intense remodeling. We investigated the density (number/area) of neurons in the DRG that were enveloped by GFAP immunoreactive SGCs. Male (N=6 per group) and female (N=6 per group) Wistar rats, 15 and 180 days old after neonatal nociceptive stimulation [1] were used. Right and left L5 DRG were removed, frozen and 8 μm thick criossections were incubated in rabbit polyclonal anti‐GFAP antibody (1:2.000; 24hr). Immunostaining protocol was used as described [2]. The DRG area was measured with the aid of computer software. Neurons enveloped by a ring of GFAP‐labeled SGCs were counted, their density (number/area) was calculated and data were compared between right and left sides, 15 and 180 days of age and genders. Differences were considered significant if p<0.05. There was an increased density of neurons enveloped by a ring of GFAP‐labeled SGCs on both genders, 15 days after the nociceptive stimulus, compared to controls, with females presenting a larger density of these neurons compared to males at this age, on the stimulated side. At age of 180 days, the difference between pain and control groups persisted only in females. The comparison between genders showed that males present a smaller density of neurons enveloped by a ring of GFAP‐labeled SGCs compared to females on both ages with statistical significance only at 15 days old groups. There is an extensive literature clearly pointing to the fact that men and women are different in their responses to pain. Women present increased sensitivity to pain and differ in pain intervention responsivity. Despite that sociocultural and psychological factors certainly play a role in these gender differences, evidence from animals studies reveal important, qualitative and quantitative differences at low levels of the neuroaxis. Our results add extra evidence that multiple biological factors and mechanisms underlie gender differences in sensitivity and responses to pain.

Support or Funding Information

Financial support: FAPESP, CNPq, CAPES and FAEPA

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Student Perceptions and Experiences with Team‐Based Learning (TBL) in a Graduate Histology Course
Authors: Azim Homaira M. , Condon Keith W. , Brokaw James J.
Published: June 2021

Contrary to the traditional didactic ways of science teaching, Team‐Based Learning (TBL) emphasizes active learning, application of knowledge, and team collaboration in the classroom. All students enrolled in a fall semester 2017 graduate histology course were invited to participate in this study. Twelve volunteer participants were asked about their opinions on TBL as well as their perceptual and experiential definition of human learning. Data collection methods included questionnaire, focus group, and direct observation. Student perceptions of and experiences with TBL were examined and linked to the literature on Tuckman's stages of group formation in order to explain some of the group dynamics in the field. Also, student perceptions on the concept of how learning occurs in general and how TBL may help in the process were examined in the context of critical constructivist theory of learning. Qualitative data analyses were performed on data across the three data sets. In general, the students perceived TBL to be a valuable learning experience. The five overarching themes identified as the requirements for a successful TBL performance on the student part were: time management, teamwork, self‐directed learning, intrinsic motivation, and learning not to resist active learning. Key significance of the TBL model were found to be in: active learning, teamwork, higher order application of knowledge, and providing formative assessment opportunities. Students' suggestions on what a good science teacher should look like and how they may avoid authoritarian exercises of power in day‐to‐day classroom activities are also documented and discussed.

Support or Funding Information

None.

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‐cell Resolution of Developing Tissues and Cell Identity Programming
Authors: Wold Barbara J.
Published: June 2021

We are using multiple single‐cell methods and platforms to deconvolve embryo limb histogenesis, tracing cell type specification and differentiation of major resident and immigrating cell lineages. We also experimentally reprogram the identity networks of closely and distantly related starting cell types, combining and comparing results from RNA‐seq and seq‐FISH data. From the resulting data, we assemble cell comprehensive identity networks. We find substantial differences in expression‐signature saliencies in developing embryo systems that have implications for future single‐cell data uses and for the design of larger scale sc‐RNA atlases. Our embryo data are part of the ENCODE consortium project, and integration of the single cell RNA‐seq data with the companion ENCODE whole‐tissue atlas, including epigenetic data, are discussed.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Multimodal single mouse and human cell ‘Omics: Is variability distinct across cellular modalities?
Authors: Eberwine James H.
Published: June 2021

With the advent of single cell analyses many unanticipated cell biological phenomena have been discovered including, new classes of RNA, identification of RNA targeting sequences and perhaps most surprisingly the discovery of cell‐to‐cell variation in transcriptome abundances even in presumptively identical cells. There is large‐scale single cell RNA variability for different cell types that can't be explained as simple molecular or technical noise. These data have led to the hypothesis that there is a many‐to‐one relationship between transcriptome states and a cell's phenotype. In this relationship the functional molecular ratios of the RNA are determined by the cell systems' stoichiometric constraints, which underdetermine the transcriptome state. Because a broad set of multi‐genic combinations support a particular phenotype, changes in the transcriptome state do not necessarily lead to changes in the phenotype. By analogy cellular phenotype should not be defined based upon the expression of individual RNAs but rather as dynamical changes in subsets of RNAs comprising selected RNA systems where the system‐associated RNAs are balanced with each other to produce the associated cellular function. Data to support these observations will be presented from live mouse and human cells, fixed tissue analysis and subcellular regional analysis. This idea provides a framework for understanding cellular heterogeneity in phenotypic response to various stimuli and provides a means for rethinking how to manipulate cellular responses so that desired outcomes are obtained including identification of molecules showing therapeutic efficacy.

Support or Funding Information

NIH U01MH098953, NIH GM110005, NIH MH 110180, The Brain Research 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.
Exploring the biosynthesis of hypermodified bases one step at a time
Authors: Bandarian Vahe
Published: June 2021

Transfer RNA (tRNA) is among the most highly modified biological molecules. To date, over 100 modifications have been documented in tRNA. Many are simple modifications, but a subset are hypermodifications that are introduced by complex, and in some cases, unknown enzymatic transformations. Our lab has been involved in deciphering the biosynthesis of queuosine and wybutosine, which are two broadly conserved hypermodified bases. This presentation will focus on recent biochemical and structural studies of the enzymes that catalyze key transformations in the biogenesis of these bases.

Support or Funding Information

NIH GM72623

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
New technologies to interrogate G protein‐coupled receptor signaling
Authors: Kruse Andrew
Published: June 2021

G protein‐coupled receptors (GPCRs) are the largest family of cell‐surface receptors in humans, and they play critical roles in processes ranging from neurotransmission to the maintenance of metabolic homeostasis. In order to better understand the molecular basis for GPCR responses to endogenous activators and synthetic drugs, we have developed a new method to track GPCR signaling in live cells using time‐resolved proximity labeling (GPCR‐APEX). This method reveals the collection of proteins a GPCR encounters following activation, and enables quantitative measurement of receptor fate following agonist stimulation. In addition to tracking GPCR signaling, we have developed new methods for the discovery of synthetic antibody fragments as modulators of receptor conformational change and signal transduction. Together with GPCR‐APEX, synthetic antibody fragment modulators allow investigation and control of GPCR signaling in new ways.

Support or Funding Information

This work was supported by grants from the Smith Family Foundation, The Vallee Foundation, and an NIH Early Independence Award (5DP5OD021345).

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 Functional Role of Arginase 1 and Neutrophil Proteomics in Predicting Ischemic Stroke Outcome
Authors: Petrone Ashley Brooke , Steele Kelsey , Eisenman Rhae
Published: June 2021

Introduction

Peripheral neutrophil (PMN) count is elevated within the first few hours of acute ischemic stroke (AIS), and neutrophilia is associated with greater AIS severity, larger infarct volume, and poor functional outcome. In animal models, PMN inhibition results in decreased infarct volume and improved functional outcome; however, this strategy has not been successfully translated in clinical trials. We hypothesize that PMNs are a heterogeneous population, consisting of both “beneficial” and “detrimental” subtypes, depending on their functional protein expression, and one PMN protein, arginase 1 (ARG1), may be a key contributor to detrimental PMN function post‐AIS. We have previously described a distinct temporal pattern of PMN expression following AIS between favorable and poor outcome AIS patients. Thus, the purpose of this study was to expand upon this examine the function relationship between ARG1, as well as other PMN proteins, and outcome following AIS.

Hypothesis

We hypothesized that PMNs isolated from AIS patients with poor outcome will have higher ARG1 expression compared to neutrophils in the favorable outcome group. Further, we hypothesize that PMNs isolated from AIS patients with poor outcome will have a distinct functional proteome, giving rise to altered or more aggressive function that leads to a poor outcome following AIS.

Methods

We quantified PMN ARG1 expression in a subset of 24 AIS patients – n=12 favorable and n=12 poor outcome. PMN ARG1 protein expression was determined by flow cytometry by co‐staining for the surface marker for PMNs, CD66b, and intracellular ARG1. Further, in a subset of these patients – n=3 favorable and n=3 poor outcome, we performed a proteomic analysis on isolated PMNs.

Results

ARG1 protein expression was significantly higher in PMNs isolated from AIS patients with a poor outcome (mean ARG1 = 665 + 117) compared to AIS patients with a favorable outcome (mean ARG1 = 453+108) (p=0.008). Further, in the samples sent for proteomics, we observed several differences in the PMN proteome between the AIS outcome groups. Specifically, the PMN proteins ‐ ARG1, defensins 1 and 4, and protein s100a9 – were increased in the poor outcome compared to the favorable outcome group.

Conclusion

In conclusion, targeting specific PMN proteins, such as ARG1, rather inhibiting PMNs as a whole, may represent a more targeted and successful approach to modulating PMNs post‐AIS to improve outcome.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pancreatic Neuroendocrine Tumors Require Homeostatic Signaling from the Unfolded Protein Response.
Authors: Oakes Scott A. , Moore Paul C. , Qi Jenny Y. , Warren Rachel , Thamsen Maike , Ghosh Rajarshi , Gliedt Micah J. , Hiniker Anne , Kim Grace E. , Maly Dustin J. , Backes Bradley J. , Papa Feroz R.
Published: June 2021

The unfolded protein response (UPR) is an intracellular signaling pathway largely controlled by two ER transmembrane kinases—IRE1alpha and PERK—that communicate the protein folding status of the endoplasmic reticulum (ER) to the nucleus to maintain ER homeostasis. Hypoxia, nutrient deprivation, proteasome dysfunction, sustained demands on the secretory pathway or somatic mutations in its client proteins‐‐conditions often encountered by cancer cells—can lead to the accumulation of misfolded proteins in the ER and cause “ER stress.” Under remediable levels of ER stress, the UPR activates transcriptional and translational changes that promote adaptation (Homeostatic UPR). But when confronted with irremediable levels of ER stress, these adaptive measures fail, and the UPR instead switches strategies to trigger cell death (Terminal UPR).

For the reasons mentioned above, ER stress is documented in many solid cancers, but whether ongoing UPR signaling is beneficial or detrimental to tumor growth remains hotly debated. Given their high secretory activity, we predicted that pancreatic neuroendocrine tumors (PanNETs) would be one neoplasm that is particularly sensitive to protein folding stress. Not only do PanNETs universally hypersecrete one more peptide hormone(s), but the endocrine cells of the pancreas from which these tumors derive are the cells in the body most impacted by genetic loss of the UPR in mice and humans. For the over 1,500 Americans diagnosed with a PanNET each year, surgery is the only potentially curative treatment. Unfortunately, the five year survival is extremely low for the ~25% of patients who develop metastatic disease. Hence, new targets are desperately needed for patients with advanced PanNETs.

We find that the UPR is strongly unregulated in human PanNETs, and that these neoplasms are heavily reliant on elevated levels of Homeostatic UPR signaling to avoid the toxic effects of protein folding stress in vivo. Moreover, we discovered that targeted interventions to either reduce Homeostatic UPR outputs or alternatively trigger the Terminal UPR have potent antitumor effects in at least 2 distinct preclinical PanNET models. Inhibiting IRE1alpha or PERK leads to hyperactivation of the opposing arm and pushes it into a Terminal UPR, resulting in the observed anti‐PanNET effects in vivo. Together, these findings suggest that the UPR is a promising target in PanNETs and related neoplasms.

Support or Funding Information

This work was supported by the National Institutes of Health: R01DK095306 (S.A.O., F.R.P), R01CA219815 (S.A.O.), R01EY027810 (S.A.O., F.R.P.), and The Neuroendocrine Tumor Research Foundation (S.A.O.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification and Characterization of TGF‐beta induced Long Noncoding RNAs in Lung Cancer
Authors: Lai Tsung‐Ching , Hsiao Michael
Published: June 2021

Lung cancer is the most lethal cancer worldwide due to high ratio of recurrence and high probability to undergo metastasis. As Epithelial‐mesenchymal transition (EMT) plays a major role during metastasis, how EMT is regulated by periphery in the lung cancer is particularly important. Although initiation of EMT can be triggered by TGF‐beta found to be overexpressed in lung cancer, how EMT can be affected by TGF‐beta signaling in different disease stages and its contribution to the heterogenecity of tumor cells are still unclear. This hampers our ability to effectively treat and block metastasis in lung cancer. Recently, long noncoding RNAs have emerged as a new player for tumor metastasis. We therefore hypothesize that lncRNAs mediated TGF‐beta signaling might be involved during EMT in lung cancer. Using RNA‐seq, we identified changes in lncRNAs profiles during different time points after TGF‐beta treatment in A549 cells. Early responded lncRNAs are major regulators in signaling and will be selected as primary candidates for further study. Expression patterns will be re‐validated by other lung cancer cell line with TGF‐beta treatment. The lncRNA candidates that have altered expression levels in response to TGF‐beta will be chosen to determine their effects on migration and gene regulation using wound healing and expression microarray. In situ hybridization will eventually be carried out to correlate the expression level of lncRNA in lung cancer clinical samples and re‐probe with EMT markers. The regulation of lncRNA in EMT would be validated with lncRNA knockout with TGF‐beta stimulation in A549 cells. The abilities of migration and invasion in knockout cells would be determined in vivo. In the end, we found the candidate lncRNA play a critical role in EMT with TGF‐beta stimulation in lung cancer.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Mechanisms of Beta Cell Adaptation to Hyperlipidemia
Authors: Poffenbarger Mason C. , Lathen Daniel R. , Ricks H. Austin , Barlow Andrew M. , Roth Zoey F. , Bikman Benjamin T. , Tessem Jeffery Sivert
Published: June 2021

Type 2 diabetes is characterized by hyperglycemia, hyperlipidemia and beta cell failure. Studies have shown that beta cell are extremely sensitive to hyperlipidemia, showing that short term cultures with elevated concentration of palmitate results in high levels of cell death. Though these studies demonstrate the toxic nature of palmitate on the beta cell, they do not accurately model the environment in which beta cells are exposed to elevated free fatty acids. We have, therefore, cultured the 832/13 beta cell line in progressively increasing levels of palmitate to determine the physiological and molecular changes that occur as beta cells adapt to this toxic environment. While 832/13 beta cells cultured immediately in 0.5mM palmitate result in sever cell death and failure to grow, 832/13 beta cells that are cultured in progressively increasing levels of palmitate (at 0.15mM, 0.3mM and 0.5mM palmitate) shows improved, although still impeded, proliferation rates. Culture in the progressively increasing concentrations causes downregulation of the beta cell transcription factors Nkx6.1 and Sox9, the glucose transporter Glut2, and the orphan nuclear receptors Nr4a1 and Nr4a3. Furthermore, as 832/13 beta cells are cultured for prolonged periods in elevated palmitate we observe downregulation of Nr4a target genes involved in mitochondrial respiration such as Idh3a, Idh3b, Idh3g, Dld and Dst. These changes correspond with modulation of mitochondrial respiration, and progressive deterioration of glucose stimulated insulin secretion, as well as decreased insulin content. These data begin to define the molecular changes that occur to the pancreatic beta cell as it is exposed to progressively increasing concentrations of palmitate, as is observed in type 2 diabetes disease progression.

Support or Funding Information

This study was funded by a BYU ORCA Grants to DRL, MCP, HAR and AMB. This study was also funded by a BYU MEG Grant to JST.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Epicatechin and its Gut Metabolites on Beta Cell Function, Survival and Proliferation
Authors: Tessem Jeffery Sivert , Rowley Thomas J. , Ray Jason D. , Herring Jacob A. , Kener Kyle B. , Bitner Benjamin F. , Ross Mimi , Lopez Moroni , Bikman Benjamin T. , Neilson Andrew P.
Published: June 2021

An initiating factor of Type 2 diabetes (T2D) is beta cell dysfunction and the eventual loss of functional beta cell mass. Mechanisms that improve beta cell function could be used as a treatment for the millions of individuals with T2D. We have recently shown that the monomeric cocoa epicatechin is sufficient to enhance insulin secretion from the 832/13 INS‐1 derived beta cell line and primary rat islets through upregulating the Nrf2 pathway, enhancing mitochondrial protein expression, improving mitochondrial function and ultimately enhancing beta cell ATP production. Here we present data pertaining to the larger epicatechin forms (oligomeric and polymeric epicatechin) as well as gut microbe metabolites of epicatechin (Homovanillic Acid, Hippuric Acid, 5‐phenylvaleric Acid) on beta cell proliferation, survival and insulin secretion. While the monomeric form enhances insulin secretion, the oligomeric and polymeric forms inhibit insulin secretion. Interestingly, the gut metabolites of epicatechin all significantly enhance insulin secretion. These results suggest that cocoa derived monomeric epicatechin and its gut‐derived metabolites could be leveraged to enhance insulin secretion as a treatment modality for patients with T2D.

Support or Funding Information

This research has been funded by a grant to JST from the Diabetes Action Research and Education 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.
Inflammatory Mediators and Fibroblasts
Authors: James La’ Toya , Reaves Titus A.
Published: June 2021

Fibroblasts are mesenchymal cells that produce and release extracellular matrix proteins such as collagens, laminins and proteoglycans during both tissue development and in response to tissue injury. In terms of tissue repair following injury, excessive healing of the injury can lead to tissue scarring. Fibroblasts have also been shown to affect neighboring cells through the release of cytokines, growth factors, and/or differentiation factors. Despite this information, little is understood about the fibroblast response to diseases characterized by acute inflammation. Necrotizing enterocolitis (NEC), is a devastating disease in response to microbial infiltration which mostly affects the gastrointestinal tract of premature infants. NEC results in nearly 2% of deaths of newborns annually. Survivors of NEC display excessive scarring in the intima of the intestinal epithelium and develop strictures leading to intestinal blockage. To that end, in the current study, we examined the fibroblast response following exposure to the following inflammatory cytokines, interferon gamma (INF‐γ), interleukin‐6, interleukin‐8 and the anti‐inflammatory cytokine, interleukin‐10. Additional inflammatory mediators Nα‐Formyl‐L‐methionyl‐phenyalanine (fMLP), Lipopolysaccharides ((LPS), 4524 [E. coli], 2515 [E. coli], Salmonella minnesota) were also exposed to fibroblasts. Fibroblast morphology shows that 4524 resulted in substantial growth with cells displaying a spindle shape while exposure to INF‐γ resulted in reduced growth with cells displaying less of the spindle morphology, but with enlarged nuclei. Other inflammatory mediators displayed minimal differences. We examined CD36 in fibroblasts exposed to 4524 and INF‐γ. Expression was reduced in INF‐γ‐exposed cells suggesting that CD36 may be involved in cellular over‐growth of fibroblasts exposed to LPS. Taken together, excessive growth and activation of fibroblasts may contribute to acute inflammatory conditions like NEC.

Support or Funding Information

NSF RII

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NON PARENCHYMAL WNTS REGULATE BETA‐CATENIN SIGNALING IN MURINE LIVER ZONATION AND REGENERATION
Authors: Preziosi Morgan , Okabe Hirohisa , Poddar Minakshi , Singh Sucha , Monga Satdarshan
Published: June 2021

β‐Catenin in hepatocytes, under the control of Wnts, regulates pericentral gene expression and contributes to liver regeneration (LR) after partial hepatectomy (PH) by regulating cyclin‐D1 gene expression as shown in b‐catenin and Wnt co‐receptors LRP5‐6 conditional knockouts (KO). However, conditional deletion of Wntless (Wls), required for Wnt secretion, in hepatocytes, cholangiocytes, or macrophages lacked any impact on zonation, while Wls deletion in macrophages only marginally affected LR. Here, we address the contribution of hepatic endothelial cells (EC) in zonation and LR by characterizing EC‐Wls‐KO generated by interbreeding Wls‐floxed and Lyve1‐cre mice. While Lyve1 expression in adult liver was limited to sinusoidal EC only, Lyve1‐cre mice bred to ROSA26‐Stopflox/flox‐EYFP mice showed EYFP labeling in sinusoidal and central vein EC. EC‐Wls‐KO mice showed notable decreases in liver weights; lacked glutamine synthetase, Cyp2e1 and Cyp1a2 in pericentral hepatocytes; and were resistant to acetaminophen‐induced liver injury. After PH, EC‐Wls‐KO failed to optimally induce cyclin‐D1 expression at 24–48 hours, which led to lower hepatocyte proliferation at 48 hours, and rebound by 72 hours. EC and macrophages isolated from regenerating livers at 12 hours showed significant upregulation of Wnt2 and Wnt9b mRNA expression, the same 2 Wnts involved in baseline b‐catenin activity in pericentral hepatocytes. Further, we show shear stress can induce Wnt2 and Wnt9b mRNA production in endothelial cells in vitro, suggesting an initiating mechanism after partial hepatectomy. In conclusion, our studies validate that at baseline, EC secrete Wnt proteins essential for b‐catenin activation in pericentral hepatocytes. During LR, primarily EC and secondarily macrophages, secrete Wnt2 and Wnt9b, to spatiotemporally regulate β‐catenin activation and cyclin‐D1 expression in hepatocytes to induce cell proliferation.

Support or Funding Information

This work was supported by NIH grants 1R01DK62277, 1R01DK100287, R01CA204586 and Endowed Chair for Experimental Pathology (S.P.M.) and NIH grant F31CA216994 (M.E.P.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Painting Chromatin with Synthetic Protein Chemistry
Authors: Muir Tom W.
Published: June 2021

Understanding protein function is at the heart of experimental biology. Perhaps one of grandest contemporary challenges in this area is to catalogue and then functionally characterize protein posttranslational modifications (PTMs). Modern analytical techniques reveal that most, if not all, proteins are modified at some point; it is nature's way of imposing functional diversity on a polypeptide chain. Understanding the structural and functional consequences of all these PTMs is a devilishly hard problem. While standard molecular biology methods are of limited utility in this regard, modern protein chemistry has provided powerful methods that allow the detailed interrogation of protein PTMs. In this lecture I will highlight how these biochemical and cell‐based tools can be used to probe a series of problems in chromatin biology.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Redefining Druggability using Chemoproteomic Platforms
Authors: Nomura Daniel
Published: June 2021

The Nomura Research Group is focused on developing and applying chemoproteomic platforms to drug the undruggable proteome to develop next‐generation disease therapies. One of the greatest challenges that we face in discovering new disease therapies is that most proteins are considered “undruggable” or difficult to target with small‐molecules. Our research group addresses this challenge by developing chemoproteomic platforms to discover and pharmacologically target unique and novel therapeutic druggable hotspots for disease therapy. We currently have four major research directions. Our first research area focuses on coupling phenotypic screening of fragment‐based covalent ligand libraries with chemoproteomic platforms to rapidly discover novel druggable hotspots, targets, and small‐molecule leads for disease therapy. Our second research area focuses on covalent ligand discovery against druggable hotspots targeted by covalently‐acting natural products that show therapeutic activity using chemoproteomic platforms to discover new therapeutic targets and synthetically tractable therapies for cancer. Our third research area focuses on advancing chemoproteomic technologies to map and pharmacologically target druggable hotspots for targeted protein degradation. Our fourth research area focuses on using chemoproteomic platforms to map on and off‐targets of environmental and pharmaceutical chemicals towards discovering new toxicological mechanisms. Our fifth research area focuses on using chemoproteomic and metabolomic platforms to discover unique and novel metabolic drivers of cancer. The talk will highlight key examples of these themes within our lab.

Support or Funding Information

National Institutes of Health (R01CA172667, P42ES004705) and Novartis Institutes for BioMedical Research

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Heme Sensor Proteins for Nitric Oxide: A Versatile Use of Captive Iron
Authors: Marletta Michael A.
Published: June 2021

Since the discovery of nitric oxide (NO) formation in animals, it has become clear that this toxic, free radical, diatomic gas plays a central role in cellular function. NO is a signaling agent in the cardiovascular system and other tissues where the NO receptor is the soluble isoform of guanylate cyclase (sGC). The immune system also utilizes NO in the host response to infection. NO in signaling is synthesized by constitutive isoforms of enzyme nitric oxide synthase (NOS) that tightly regulates NO formation; thereby avoiding toxicity. sGC contains a heme cofactor that traps NO, thereby activating the enzyme to convert GTP to cGMP. The heme domain of sGC was found to be part of the H‐NOX (Heme‐Nitric oxide OXygen) family of proteins with homologues in aerobic and anaerobic prokaryotes. Some H‐NOX domains form stable and tight complexes with oxygen while others are selective for NO. Structural and biochemical studies have provided a molecular explanation for selective NO trapping at low concentrations. Study of H‐NOX sensors has uncovered novel functions in biology such as oxygen‐regulated sGCs and NO sensors in pathogens. The function of NO in biology is even more pervasive and more complicated than originally described. The structure/function picture that is emerging points to some common themes though many questions remain.

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