bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cell Cycle Regulation and Hypoxic Adaptation in the Pathogenic Yeast Cryptococcus neoformans
Authors: Kawamoto Susumu , Moranova Zuzana , Virtudazo Eric , Ohusu Misako , Raclavsky Vladislav 
Published: June 2021

We have reported that the cell cycle behavior of the pathogenic yeast Cryptococcus neoformans (C. neoformans) is different from the cell cycle control exhibited by the model yeast Saccharomyces cerevisiae, and also have reported the molecular characterization and physiological roles of the two main eukaryotic cell cycle genes, C. neoformans cyclin dependent kinase 1 (CnCdk1) and cyclin homologues. We also identified components of the G2‐M control machinery, Wee1 kinase and Cdc25 phosphatase homologues, in C. neoformans. Wee1 and Cdc25 are involved in the inhibition and activation, respectively, of the Cdk1‐cyclin complex at the mitotic entry checkpoint in eukaryotes.On the other hand, in the obligate aerobic pathogenic yeast C. neoformans, limited aeration has been demonstrated to cause slowdown in proliferation and delayed budding, resulting eventually in a unique unbudded G2‐arrest. The ability to adapt to decreased oxygen levels during pathogenesis has been identified as a virulence factor in C. neoformans. We have identified and characterized the gene that is necessary for the proliferation slowdown and G2‐arrest caused by limited aeration. Random mutants were prepared and screened for lack of typical slowdown of proliferation under limited aeration. The gene coding for a zinc‐finger transcription factor was identified in mutants showing most distinctive phenotype. Targeted deletion strain and reconstituted strain were prepared to characterize and confirm the gene functions. This gene was also identified in a parallel studies as homologous both to calcineurin responsive (Crz1) and PKC1‐dependent (SP1‐like) transcription factors. We have confirmed the role of the cryptococcal homologue of CRZ1/SP1‐like transcription factor in cell integrity, and newly demonstrated its role in slowdown of proliferation and survival under reduced aeration, in biofilm formation and in susceptibility to fluconazole. Our data demonstrate a tight molecular link between slowdown of proliferation during hypoxic adaptation and maintenance of cell integrity in C. neoformans and present a new role for the CRZ1 family of transcription factors in fungi. The exact positioning of this protein in cryptococcal signalling cascades remains to be clarified.Support or Funding InformationThis work was partially supported by Grants‐in‐Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan and Cooperative Research Program of Medical Mycology Research Center, Chiba University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of Autophagy by Mevalonate Pathway Inhibitors, a New Therapeutic Approach to sensitize Glioblastoma Cells to Temozolomide Induced Apoptosis
Authors: Shojaei Shahla , Alizadeh Javad , Thliveris James , Koleini Navid , Kardami Elissavet , Hatch Grant , Xu FY , Hombach‐Klonisch Sabine , Klonisch Thomas , Ghavami Saeid 
Published: June 2021

Glioblastoma multiforme (GBM) is the deadliest brain tumor with an approximate 20 month survival rate after diagnosis and treatment. Temozolomide (TMZ), the chemotherapeutic drug of choice for GBM, is an alkylating agent that causes DNA damage. TMZ treatment results in the induction of apoptosis in GBM cells, however, it induces autophagy and consequently chemoresistance. Statins are mevalonate (MEV) cascade inhibitors with beneficial effects on the enhancement of the survival rate of patients with different types of cancer, particulary if consumed long term before diagnosis of cancer. Here, we determined the effect of simvastatin (Simva), a blood brain barrier permeable statin, on the sensitization of GBM cells to TMZ induced apoptosis through inhibition of autophagy flux. We pretreated two GMB cell lines, U251 and U87 cells, with low doses of Simva (1 and 2.5 μM, respectively) with or without different intermediates of the mevalonate cascade: MEV (2.5 mM), Farnesyl pyrophosphate (FPP) (15 μM), Geranylgeranyl pyrophosphate (GGPP) (15 μM) and cholesterol (50 μM) for 4 h and then treated cells with TMZ (100 μM) for 48–96 h. A signficiantly reduced viability and increase in the population of apoptotic dead cells were observed in GBM cells treated with the combination of Simva‐TMZ compared to cells treated with TMZ alone. Addition of MEV, FPP, GGPP and cholesterol did not attenuate these effects significantly. Sima‐TMZ treatment did not alter the total cholesterol pool in U87 and U251 cells compared to controls. Western blot analysis, immunocytochemistry and transmission electron microscopy revealed that the combination of Simva with TMZ inhibited autophagic flux. Overall, the results suggest that sensitization of GBM cells to TMZ‐induced apoptosis by Simva is independent on the cholesterol biosynthetic pathway but may involve inhibition of autophagy flux.Support or Funding InformationSupported by funding from a Mitacs Accelerate PDF Award & a Health Science Center Foundation Operating 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.
Muscarinic Antagonist Mediated Activation of ERK Signaling Depends on β‐arrestin Recruitment to Augment Axonal Outgrowth in Neurons
Authors: Sabbir Mohammad Golam , Fernyhough Paul 
Published: June 2021

ObjectiveOne of the major cellular effectors activated by G protein coupled receptors is extracellular signal‐regulated kinase (ERK). The ERK signaling cascade regulates a variety of cellular processes including growth and proliferation. Both G protein and β‐arrestin mediated signaling pathways lead to ERK activation by phosphorylation through different kinases. Recently, we have shown muscarinic acetylcholine type 1 receptor (M1R) antagonists, muscarinic toxin 7 (MT7) and pirenzepine, induced elevated neurite outgrowth and protected from small and large fiber neuropathy in adult sensory neurons in various animal models. Thus, we hypothesized that the neuritogenic effect of muscarinic antagonists was mediated by M1R‐ERK signaling.MethodologyWe have used extensive two dimensional isoelectric focusing/SDS‐PAGE combined with analysis using multiple phospho‐epitope specific antibodies to study ERK1/2 phosphorylation and activation of its downstream nuclear effector ‐ cyclic response element binding protein (CREB) in cultured adult rat primary dorsal root ganglion (DRG) neurons. Activation of CREB is known for neuroprotective and growth promoting effects. In addition, we have used CRISPR/Cas9 based β‐arrestin1/2 and GαS/L/Q/11/12/13 null HEK293 cell lines to dissect the downstream signaling events following antagonist binding to M1R.ResultsMT7 and pirenzepine‐mediated M1R‐ERK signaling induced a significant increase in activation‐loop specific phosphorylation (T202/Y204) of ERK1/2 and significantly elevated pCREB(S133) levels in cultured sensory neurons. Further, using M1R expressing β‐arrestin1/2 and GαS/L/Q/11/12/13 null HEK293 cell lines, we have shown that MT7 or pirenzepine mediated ERK1/2 activation is positively dependent on β‐arrestin recruitment to M1R but negatively regulated by G proteins. We have identified multiple charged fractions of ERK1/2 modulated by muscarinic antagonist treatment that is indicative of complex dynamics related to allosteric drug binding.ConclusionsOur study provides evidence that the neuritogenic effect of MT7 and pirenzepine may be mediated by selective blockade of M1R and downstream activation of ERK‐CREB signaling. We also give insights into allosteric drug binding and complex dynamics of ERK activation. In addition, our study also indicates that removal of negative regulation by G proteins on antagonist‐M1R‐ERK signaling could be exploited for improved therapy in neuropathic diseases.Support or Funding InformationSupported by CIHR grant # MOP‐130282 (PF)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of the putative GTPase activity of the Ellis van Creveld protein 1
Authors: Nguyen Giang T. , Odunuga Odutayo O. 
Published: June 2021

Ellis van Creveld (EvC) syndrome is a genetic disorder characterized by mild short stature, postaxial polydactyly, dysplastic nails and teeth. Loss‐of‐function EVC1 and EVC2 proteins resulting from mutations in EVC1 and EVC2 genes, respectively, have been identified as the primary causes of EvC syndrome. Essential to their function is the physical interaction of EVC1 and EVC2 proteins at the base of the primary cilium, where this complex regulates the Sonic and Indian hedgehog (Hh) signaling systems. Recent studies have revealed that this EVC1‐EVC2 protein complex is tethered to the base of the primary cilium by another complex comprising of two newly identified proteins, EFCAB7 and IQCE. Together, this multiprotein complex positively regulates downstream processes after signaling activation by hedgehog proteins. The presence of a putative P‐loop sequence in EVC1 strongly suggests that its interaction with EVC2 is dependent on nucleotide hydrolysis. Using protein fragments, we have experimental evidence that suggest that EVC1 possesses an intrinsic GTPase activity conferred by the presence of the P‐loop. This observation may provide insight into the function and regulation of the EVC1‐EVC2 complex in hedgehog signaling pathways.Support or Funding InformationWelch Foundation Grant # AN‐0008FRG‐RCA Grant, SFASUThis 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 Interaction between Glucocorticoid Receptor and beta‐arrestin proteins
Authors: Petrillo Maria Grazia , Cidlowski John 
Published: June 2021

Glucocorticoids (GCs), vital stress hormones released as the end products of the hypothalamic‐pituitary‐adrenal axis, are involved in controlling inflammation. The actions of glucocorticoids occur through the activation of the glucocorticoid receptor (GR) which, like other members of the steroid hormone receptor superfamily, functions as a ligand‐activated transcriptional regulator. Throughout its intracellular journey, liganded GR creates physical contacts with regulatory proteins, thus affecting its activity and function. The protein‐protein interactions of GR are decisive for the resolution of the glucocorticoid‐dependent physiological processes. We have recently discovered a new interaction between GR and beta‐arrestin‐1 (barr‐1). Beta‐arrestin‐1 plays a well‐established role in participating in agonist‐mediated desensitization of G‐protein‐coupled receptors (GPCRs). However, it has become increasingly appreciated as a scaffold protein, thus conferring novel signaling properties independent of GPCR activity. These functions include interfering with the ubiquitin‐proteasome machinery. Moreover, recent studies, from our lab, have demonstrated that glucocorticoids can promote the transcription of beta‐arrestin‐1, thereby contributing to modulating its function. These findings led us to investigate the possibility that barr‐1 and GR can either form a tightly regulated loop, or that barr‐1 may contribute to the activity of the GR. To investigate whether the association of barr‐1 with GR affects the activity of GR, RNA sequencing in control‐ and barr‐1 knock down‐ A549 cell line has been performed. The transcriptome analysis identified post‐translational modifications as one of the top‐ranked cellular functions significantly altered by the lack of barr‐1. Notably, lack of barr‐1 reshaped the GR gene signature, regulating a cohort of genes responsible for the activation of the ubiquitin‐proteasome machinery. Among these genes, PELI1 (Pellino E3 Ubiquitin Protein Ligase 1) resulted the most upregulated glucocorticoid‐responsive gene. Indeed, in vitro data demonstrated that when beta‐arrestin‐1 was knocked down, it accelerated GC‐induced GR degradation reducing its half‐life and promoting its ubiquitination via PELI1 activation. These observations strongly suggest that barr‐1 is required for regulating GR expression, by limiting proteasomal degradation of GR. Playing a pivotal role in homeostasis, GR is susceptible to numerous stimuli that modulate its activity, and discovering a novel protein partnership between GR and barr‐1 may help in developing new glucocorticoids, as well as combination therapies that sustain the beneficial effects of glucocorticoids limiting the risks.Support or Funding InformationThe research project is supported by the Intramural Research Program of the NIEHS.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ALLOSTERIC MODULATION OF THE PENTAMERIC LIGAND‐GATED ION CHANNEL ELIC BY FUNCTIONALLY ACTIVE NANOBODIES
Authors: Ulens Chris , Brams Marijke , De Peuter Hannelore , Spurny Radovan , Pardon Els , Bertrand Daniel , Steyaert Jan , Govaerts Cedric 
Published: June 2021

Pentameric ligand‐gated ion channels (pLGICs) or Cys‐loop receptors are a class of ion channels involved in fast synaptic transmission in the central and peripheral nervous systems. Members of this family include the nicotinic acetylcholine receptors, serotonin type‐3 receptors, GABAA/C (gamma‐aminobutyric acid) receptors and glycine receptors. These receptors are the target for a wide variety of therapeutics, including benzodiazepines such as diazepam (Valium), which act as positive allosteric modulators (PAMs) of the GABAA receptor and are prescribed as a sedative, anxiolytic or anti‐epileptic. In this study, we employed the prokaryote ligand‐gated ion channel ELIC as a model to understand the structural mechanism of allosteric modulation. We raised nanobodies, which are antibody fragments derived from camelids and have the potential to trap conformationally transient states, and used them as crystallization chaperones for ELIC. Functional characterization of ELIC nanobodies using electrophysiological techniques demonstrated that these nanobodies are active as channel modulators. We demonstrate that nanobodies can either inhibit or potentiate agonist‐evoked channel responses, suggesting that nanobodies can act as negative (NAMs) or positive allosteric modulators (PAMs), respectively. The X‐ray crystal structure of ELIC in complex with a nanobody reveals that a nanobody binds at an allosteric binding site and near to a binding site previously known to mediate potentiation of ELIC by the benzodiazepine flurazepam. Given the structural conservation of allosteric binding sites between ELIC and human receptors our study reveals common mechanisms for allosteric modulation in this family of channels. Our expanded knowledge of the structural determinants of allosteric modulation opens opportunities for the development of new therapeutics targeting pLGICs in ion channel‐related disorders.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Adaptation to Supraphysiologic Room Air Oxygen Changes the Effects of Nitric Oxide on A549 Lung Cells In Vitro
Authors: Henn Alicia , Darou Shannon , Yerden Randy 
Published: June 2021

Airway endothelial cells are exposed to some of the highest oxygen levels encountered in vivo (~10% O2), however, these levels are significantly lower than room air (21% O2). The culture and handling of airway cells in room air oxygen incubators can change the redox status of the cells, introducing artifact. Nitric oxide, which can affect HIF‐1a stabilization and reversibly bind cytochrome oxidase, has been shown to play multiple roles in the response of lung to hypoxic injury and inflammation. Here we explore whether pre‐adaptation of the commonly used human lung epithelial cell line A549 to room air oxygen levels changes cellular responsiveness to NO. The null hypothesis was that pre‐adaptation to different oxygen levels would have no effect on responses to NO. Using the Xvivo System to provide full‐time control of oxygen and CO2 levels during cell incubation as well as handling, we adapted A549 cells for growth at 18% supraphysiologic oxygen (standard room air incubator level with humidity and CO2), physiologically relevant oxygen (10%) or pathophysiologic hypoxia (2% O2). We assayed cellular responses to gaseous nitric oxide (NO) exposure in standard plate‐based cytotoxicity and migration assays and assayed local nitric oxide levels in the cell culture media. We found that adaptation to different oxygen levels had a concentration‐dependent effect on the responses of A549 cells to gaseous NO. We concluded that it is critical to consider the oxygen‐adapted state of the cell prior to assay to avoid oxygen artifact.Support or Funding InformationAll support was from BioSpherix, Ltd.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 Moderate Voluntary Ethanol Consumption on the Rat and Human Gut Microbiome
Authors: Pinkowski Natalie J. , Kosnicki Kassi L. , Penprase Jerrold C. , Cintora Patricia , Torres Pedro J. , Harris Greg L. , Brasser Susan M. , Kelley Scott T. 
Published: June 2021

Many alcohol‐induced health complications are directly attributable to the toxicity of alcohol or its metabolites, but another potential health impact of alcohol may be on the microbial communities of the human gut. Clear distinctions between healthy and diseased‐state gut microbiota have been observed in subjects with metabolic diseases and recent studies suggest that chronic alcoholism is linked to gut microbiome dysbiosis. Here, we investigated the effects of alcohol consumption on the gut microbiome in both rats and humans. The gut microbiota of rats voluntarily consuming a 20% ethanol solution, on alternate days, were compared to a non‐exposed control group to identify differential taxonomic and functional profiles. Gut microbial diversity profiles were determined using culture‐independent amplification, next‐generation sequencing and bioinformatic analysis of bacterial 16S ribosomal RNA gene sequence libraries. Our results showed that, compared to controls, ethanol‐consuming rats experienced a significant decline in the biodiversity of their gut microbiomes, a state generally associated with dysbiosis. We also observed significant shifts in the overall diversity of the gut microbial communities and a dramatic change in the relative abundance of particular microbes, such as the Lactobacilli. We also compared our results to human fecal microbiome data collected as part of the citizen science American Gut Project. In contrast to the rat data, human drinkers had significantly higher gut microbial biodiversity than non‐drinkers. However, we also observed that microbes that differed among the human subjects displayed similar trends in the rat model, including the Lactobacilli as well as bacteria implicated in metabolic disease.Support or Funding InformationNIH AA023291This 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 cell wall perturbation reagents and plant phenolic compounds on growth, colony morphology, and germination of the corn rot fungus Fusarium verticillioides
Authors: Pierson Paige N. , Hardtke Haley , Brown Daren W. , McQuade Kristi L. 
Published: June 2021

Fusarium verticillioides is a filamentous fungus known to infect corn and produce toxins called fumonisins, resulting in crop losses around the globe. Our lab previously showed that an F. verticillioides strain unable to produce the disaccharide trehalose is not only less pathogenic to corn than the wild‐type fungus but also unable to synthesize fumonisins. We are exploring the origins of the observed reduction in pathogenicity of this trehalose‐deficient strain. To test whether cell wall integrity of this strain is compromised, we measured the effects of cell wall perturbation reagents on colony morphology, radial growth, and germination kinetics. Both Congo Red and Calcofluor‐white have a greater effect on the trehalose‐deficient strain than on wild type. Interestingly, Congo Red causes significant hyphal clumping in the trehalose‐deficient strain. A comparison of the effects of phenolic plant defense compounds (caffeic or vanillic acid) on growth of the two strains is also reported.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interdisciplinary undergraduate research program with a travel component at Mount Saint Mary's University Los Angeles to promote success of women in STEM
Authors: Nogaj Luiza A. 
Published: June 2021

In response to the national need to produce skilled and globally aware STEM students, Mount Saint Mary's University, Los Angeles developed an interdisciplinary undergraduate research program with an international fieldwork experience in the summer. The goals of the program are: (1) to train undergraduates in interdisciplinary research methodologies (laboratory techniques, cross cultural survey research, and policy analysis); (2) to collect real fieldwork data in Peru; (3) to analyze and present the results in professional conferences and publications; and (4) to develop leadership skills and teamwork necessary for student success and retention in STEM fields.Students enrolled in the program spend two semesters doing course work. In the first semester they learn about the molecular, environmental and nutritional causes of cancer from MSMU's STEM faculty and about the disparities in policy and healthcare access in the U.S. and Peru from political science faculty members. In the second semester, the students develop research skills in both STEM disciplines and the social sciences: laboratory research skills in biology and chemistry, and survey methodology in political science. The students develop an evidence gathering plan, design a survey, and seek an IRB approval in preparation for their global research experience over the summer.During the summer, the students first interview women in urban and rural areas of Peru about factors contributing to their health, such as access to healthcare and nutritional habits.During the culminating, third semester of the program, the students analyze their survey data, present their results at conferences and develop leadership skills. They also examine barriers to success for women in STEM. We expect this combination of regular course work with a summer research component, international field study, and leadership training to be an effective way to increase success and retention rates of undergraduate students in the STEM fields at MSMU.This program is a model for future research topics thus creating a sustainable and meaningful undergraduate research training.Support or Funding InformationMSMU LA Presidential FundThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.