bluuebunny/crossref_metadata_2025 huggingface dataset:
Advantages of Using Magnetic Resonance Arteriogram Images to Create 3D Virtual Anatomic Vasculature Models
Authors: Yang Gongchao , Storrs Judd , Lynch James C. , Lehman Michael N. , Cui Dongmei
Published: June 2021

Computed tomography images are commonly used in creating 3D virtual models for anatomical education. Recently, use of computed tomography arteriogram (CTA) images has been reported for construction of vascular virtual models for anatomy teaching, but small blood vessels are difficult to capture during the creation process. High contrast Magnetic Resonance Arteriogram (MRA) images offer better resolution for visualizing and capturing the small branches of the cerebral vasculature than CTA images. In order to develop better vascular models, improving the quality of models and their effectiveness as teaching tools, we used MRA Images as a source and combined the images using a new, innovative, semi‐auto‐combination technique. We used Amira software to develop 3D virtual models that can be presented stereoscopically in a virtual reality environment. The images and models are virtually displayed in stereo mode and viewed by students wearing polarized 3D glasses allowing enlargement, rotation in 360 degrees and in any plane. The stereoscopic 3D Magnetic Resonance Arteriogram models allow students to view and interact with normal and abnormal cerebral vasculature, including posterior cerebral artery hemorrhage in greater detail than has previously been possible.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Changes of the Intercondylar Notch and their impact on the morphology of the cruciate ligaments in osteoarthritis.
Authors: Hirtler Lena , Schreiner Markus , Röhrich Sebastian , Hirtler Arno , Kainberger Franz
Published: June 2021

Introduction

As the anterior cruciate ligament (ACL) is not only impinged against the roof but also against the lateral wall of the intercondylar notch (IN), information of changes during life and especially the progression of osteoarthritis could be relevant in predicting the risk for ACL‐rupture and ‐degeneration. Therefore the purpose of this study is to evaluate the influence of osteoarthritis on notch morphology, on the cruciate ligaments as well as on the meniscofemoral ligaments.

Material and Methods

Image data was retrieved from the osteoarthritis initiative (part 2). In coronal and axial images osseous structures were measured. At the level of the popliteal sulcus and on the level of the joint line, the width of the lateral and medial femoral condyle, the notch width (NW) as well as the total width of the distal femur were measured. The notch width index (NWI) was calculated. On whole‐leg radiographs the leg alignment was measured. The morphology of the ACL, the posterior cruciate ligament (PCL) and the meniscofemoral ligaments were assessed.

Results

The morphology of the IN is directly influenced by the development of osteoarthritis. Three types of shapes (A‐shape, Inverse‐U‐shape, Ω‐shape) were defined to characterize these changes. Especially the Ω‐shape is directly correlated with rupture of the ACL. Changes to the IN also significantly influence the morphology of the other ligamentous structures.

Conclusion

To reflect the intimate correlation of the osseous structures and the ligaments of the IN, the term intercondylar space was created. It is not a static area but undergoes continuous changes throughout life. At the level of the joint line a NWI <0.17 and a NW < 14 mm should be interpreted as notch stenosis and are definite risk factors for ACL rupture. Measurements combined with the evaluation of the shape of the IN could improve risk estimation for ligament pathologies through semiquantitative assessment.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hepatic Autophagy Gene Expression Is Induced by Post‐Weaning Diets in Sprague‐Dawley Rats fed with A Low‐Protein Diet During Lactation
Authors: Cai Mingzhu , Wang Huan , Chen Hong , Pan Yuan‐Xiang
Published: June 2021

Background

Both maternal dietary protein restriction and excess fat intake are associated with numerous metabolic responses including the activation of transcription factor 4 (ATF4). ATF4 is one of the master regulators of stress response pathways, which potentially program autophagy genes expression in the liver of offspring rats.

Hypothesis

The current study tested the hypothesis that lactational low‐protein (LP) diet and postnatal high‐fat (HF) diet induce a metabolic mismatch that activates autophagy gene expression through the activation of ATF4 in the liver of offspring rats.

Methods

Timed‐pregnant Sprague‐Dawley rats were fed a control diet (C, 20% protein) during gestation followed by either a LP diet (8% protein) or the control diet during lactation for 21 days. At weaning, the offspring was fed either C (16% fat) or HF (45% fat) to generate three offspring groups: C/C/C, C/LP/C and C/LP/HF. At 12 weeks of age, liver samples of male and female rats were collected for analysis.

Results

Lactational LP diet and post‐weaning HF diet induce autophagy gene expression differentially in the liver of male and female offspring. mRNA expression of Beclin1, p62/sqstm1, LC3b and Atg5, crucial markers of autophagy, was significantly upregulated by lactational LP diet (C/LP/C when compared to C/C/C, p<0.05) in female offspring, but not in male offspring. Consistent with that, mRNA expression of other autophagy related genes including Atg16l1, Atg12 Atg3, Gabarapl2, Atg7, Atg10 and Gabarap increased significantly in the female offspring only. Moreover, autophagy genes Atg16l1, Atg12, Atg3, Beclin1, p62/sqstm1, LC3b, Gabarapl2, Atg7, Atg10, Gabarap, and Atg5 were expressed at a higher level by post‐weaning HF diet (C/LP/HF when compared to C/LP/C, p<0.05) in both male and female offspring. A master regulator of stress pathway, transcription factor ATF4, also displayed differential expression in the mismatched dietary groups.

Conclusion

Lactational low‐protein diet induced autophagy related gene expression in female offspring and the induction was further augmented by a post‐weaning high‐fat diet. On the other hand, in the liver of male offspring, only post‐weaning high‐fat diet induced mRNA expression of autophagy related genes. The differential regulation of autophagy by diets, sex, and developmental periods could be explained by dysregulation of stress response pathway involving transcription factor ATF4. Overall, our data demonstrated that lactational low‐protein diet may impact autophagy pathway more in female offspring than in male offspring rats.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gene architecture influences on the outcome of INDEL‐based genome editing
Authors: Tuladhar Rubina , Yeu Yunku , Piazza John Tyler , Clemenceau Jean , Wu Xiaofeng , Tan Zhen , Mathews David , Hwang Tae Hyun , Kim James , Lum Lawrence
Published: June 2021

The introduction of insertion‐deletions (INDELs) by activation of the error‐prone non‐homologous end joining pathway forms the mechanistic basis of CRISPR/CAS9‐directed genome editing. The anticipated impact of INDELs on protein products largely is based on the presence or absence of pre‐mature termination codons in altered mRNA which in turn control non‐sense mediated decay regulation. From analyzing gene‐protein relationships in a large collection of CRISPR/CAS9 edited cell lines, we have identified mechanisms that frequently result in the production of foreign proteins and that compromises the utility of this prediction model. These mechanisms include: a) the alteration of RNA secondary structure resulting in the inadvertent introduction of internal ribosomal entry sites, b) the conversion of pseudo‐RNAs into protein encoding molecules, and c) the induction of exon skipping due to compromising exon splicing enhancers. We leveraged our new understanding of how these cellular mechanisms that ordinarily safe guard genomic integrity influence CRISPR/CAS9 genetic outcomes to systemize the re‐design of sgRNAs reagents for gene deletion strategies and for the rescue of genetic alterations associated with inherited diseases.

Support or Funding Information

Welch Foundation (I‐1665), CPRIT (RP130212), and RO1‐CA168761 and RO1C1A196851.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reversal of phenotypic abnormalities by CRISPR/Cas9‐mediated gene correction in iPSCs derived from Fabry IVS4+919 mutation patients
Authors: Song HuiYung , Chien Chien‐Shiu , Chiou Shih‐Hwa , Chien Shu
Published: June 2021

Fabry disease (FD) is the second most common lysosome storage disorder (LSD) that is caused by deficiency of the lysosomal enzyme α‐galactosidase A (GLA). More than 500 GLA mutations have been identified in FD that result in GLA enzyme deficiency and lead to lysosomal globotriaosylceramide (Gb3) accumulation. In FD patients in Taiwan, there is a high incidence of the GLA IVS4+919G>A mutation, which leads to late‐onset cardiovascular dysfunction. Crucial issues for establishing isogenic control iPSCs include the minimization of the chance of missing modifiable effects relevant to the pathogenesis and the increase of confidence in identifying FD phenotypes and mechanisms. Here we report correction of GLA IVS4+919G>A human induced pluripotent stem cells (hiPSCs) using the CRISPR/Cas9 approach. Both FD and corrected isogenic hiPSCs can be differentiated into vascular endothelium that exhibits the rich functional phenotypic plasticity of mature and functional vascular endothelium which can respond to physiological level of flow and inflammatory stimulation in a circulating flow chamber system. We demonstrated that phenotypic abnormalities in FD hiPSC‐derived endothelial cells, including intracellular Gb3 accumulation, autophagic flux impairment, and reactive oxygen species (ROS) production, are rescued in isogenic controls. Importantly, RNA‐seq profiling revealed that a number of apparent gene expression differences detected between FD and healthy control lines are absent between FD and corrected lines, indicating that these differences are likely related to genetic background rather than FD‐specific effect. Such information is highly valuable for the development of methods for the prevention and intervention of the adverse effects of FD‐associated cardiomyopathy.

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 Editing Mediates Oligomerization State of Calcium‐Dependent Activator Protein for Secretion 1 (CAPS1)
Authors: Mitchell Ben , Ulbricht Randi J.
Published: June 2021

CAPS1 RNA under‐goes a site‐specific adenosine‐to‐inosine RNA editing event that alters a genomically‐encod‐ed glutamate (GAG) to a glycine (GIG) codon within the encoded Calcium‐dependent activator protein for secretion 1 (CAPS1) protein. The protein isoforms generated from edited and non‐edited tran‐scripts have differential effects on synaptic vesicle distribution, release and recycling. Increasing expression of non‐edited CAPS1 isoforms negatively affects vesicle evoked release, leads to a more diffuse distribution of synaptic vesicles and increases spontaneous release from synaptic vesicles. Conversely, elevation of edited CAPS1 isoforms stimulates increased clustering of synaptic vesicles and decreased constitutive release from synaptic vesicles. We hypothesize that the mechanism for the distinct editing‐dependent phenotypes is related to the oligomerization state of CAPS1. To test this hypothesis, recombinant isoforms of edited and non‐edited CAPS1 were transfected into human embryonic kidney cells and detected by western blot of native PAGE. We find that non‐edited CAPS1 isoforms have a distinct oligomerization state from edited isoforms, suggesting that RNA editing influences the ability of CAPS1 to form larger complexes. Overall, we conclude that RNA editing serves as a molecular switch, allowing CAPS1 to self‐associate, thereby organizing vesicles into a tightly knit cluster that promotes robust, regu‐lat‐ed release and preventing release from vesicles in the absence of a stimulus.

Support or Funding Information

This work is supported by Missouri State University, Department of Biomedical Sciences.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating on the binding characteristics of the important RNA‐protein interactions in Brome Mosaic Virus
Authors: Riboul Rashid , Siegel Noah , Kim Chul
Published: June 2021

RNA‐protein interactions are often crucial for viability of many biological systems, including the replication cycle of RNA viruses. Understanding molecular driving forces behind the binding specificity of these interactions in RNA viruses is important in advancing our knowledge not only in the mechanism of viral replication but also in designing therapies against viral infections. Here, we have investigated the binding characteristics of the N‐terminal region of a viral Coat Protein (CP) to promoter sequences of the genomic RNA in Brome Mosaic Virus (BMV), a granary plant‐infecting RNA virus. BMV has three separate RNA molecules as its genomic RNA and coordinating the timely production of these three RNAs is crucial in successful replication of their progeny viruses. Previous studies showed that the coordinating function is conducted by the CP, which involves its specific binding on BMV RNA. The N‐terminal region of CP binds to a couple of promoter sequence of BMV genomic RNA. Interestingly those RNA motifs all have well‐defined loop structures. We have explored on the importance of the three‐dimensional conformations of these RNA motifs for the recognition by the N‐terminal region of CP. We designed peptides mimicking the N‐terminal region of CP, called CPNT, and also prepared RNA sequences representing the promoter motifs of BMV RNA. We have used Fluorescence quenching & anisotropy as well as NMR and FPLC for investigating the binding characteristics of CPNT to these RNA. Our initial results have shown that one of the RNA promoter motifs, found at the 5′end of BMV genomic RNA, showed specific binding affinity in which the conformation of its triloop (5′AUA3′) serves a key recognition motif for the CPNT‐RNA interaction. We are currently investigating the binding characteristic of other RNA motifs with CPNT as well as the effects of pH, salt, and temperature on them. Fluorescence spectroscopy and Fast Protein Liquid Chromatography are used to measure and compare binding efficiencies of peptide‐RNA complexes for pH 7.5, 7.0, 6.5, 6.0, and 5.5 in sodium chloride and ammonium acetate, with native polyacrylamide gel electrophoresis (PAGE) as a way to visualize binding of those complexes. High‐resolution 1D and 2D NMR is used to gain structural insights into complexes. These new results will be presented at the conference.

Support or Funding Information

This project was supported by the Center for Student Research and by the Faculty Support Grant from CSU East Bay. A Varian 500 MHz FT‐NMR instrument was obtained with funds from NSF MRI grant 0420615 and the CSU East Bay College of Science.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Promoting Protein Translation in a Vaccinia Virus System Using Translation Enhancing Elements
Authors: Minder Stephanie , Richard Brent , Jacobs Bertram L. , Wellensiek Brian P.
Published: June 2021

Vaccinia virus (VACV) is known for its use in the vaccination strategy that led to the eradication of smallpox, but has more recently been seen as a potential agent against other pathogens. This is due to the fact that the VACV genome can be easily manipulated to express genes from other pathogens and can infect a wide range of cell types. In exploring its use as a recombinant vaccine, VACV was attenuated to decrease the side effects associated with a wild‐type VACV infection. Through this attenuation VACV is unable to replicate efficiently within host cells. A drawback of this attenuation however, is a reduction in viral protein expression. This reduced protein production, while leading to a reduction in side effects, also results in a decreased host immune response. Therefore, making the candidate vaccine less effective. In order to address this issue, the current research is focused on amplifying recombinant protein production in an attenuated VACV model. A potentially useful tool in attempting to do this lies in human translation enhancing elements. In previous research, these elements were found to boost protein production using a cytoplasmic VACV based expression system. One specific translation enhancing element hTEE‐658 was found to increase expression 10‐fold when compared to other translation enhancing elements. Recent research in our lab has demonstrated that, although hTEE‐658 increases protein expression in an attenuated virus system, it does not increase to comparable levels driven by the wild‐type VACV. In this current study, we attempted to further increase protein expression by combining hTEE‐658 with other top performing translation enhancing elements. Analysis using luciferase reporter plasmids highlighted one combination increased protein expression when compared to hTEE‐658 alone in a wild‐type VACV based system. A similar trend observed when expression was driven with an attenuated virus. This pattern of protein expression also remained consistent when a pathogen specific gene was used in place of luciferase. This research demonstrates that combining translation enhancing elements is a viable method for increasing protein production in an attenuated VACV model, and provides an interesting avenue to increase the effectiveness of recombinant VACV vaccines.

Support or Funding Information

Biomedical Sciences Graduate Program & Midwestern University intramural funds awarded to BPW

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pentachlorophenol Effects Synthesis of IL‐1β in Human Immune Cells
Authors: Martin Tamara , Whalen Margaret
Published: June 2021

Pentachlorophenol (PCP) is an environmental contaminant found in human blood. PCP has been used in fungicides, insecticides, herbicides, and antifouling paint. A number of cancers and diseases are associated with exposure to this contaminant. Interleukin 1‐βeta (IL‐1β) is a cytokine produced by lymphocytes and monocytes. It is involved in stimulating cell proliferation, tissue repair, and present during the inflammatory response to injury. Increased levels of IL‐1 β have been associated with rheumatoid arthritis, systemic lupus erythematosus, psoriasis, ulcerative colitis, and tumor progression. A decrease in levels of IL‐1 β could lead to lack of an appropriate immune response to a pathogen while elevations in its levels may lead to increased potential for inflammatory disorders and tumors. Previous studies in our lab show that PCP increases secretion of IL1β from peripheral blood mononuclear cells (PBMCS) and monocyte‐depleted PBMCS. Increased secretion could be due to PCP‐induced release of a store of pre‐existing cytokine in these cells or due to PCP‐induced increases in the synthesis of IL‐1β. The objective of this study was to examine if exposure of human immune cells to PCP increases the synthesis (secreted + intracellular levels) of IL1β. PBMCS were exposed to 5 μM to 0.05 μM PCP for 10 min, 1 h, 6 h, and 24 h. PBMCs showed increased synthesis of IL1β after 6 h and 24 h exposures to PCP. The concentrations of PCP at which these increases occurred varied among the donors. These results suggest that PCP‐induces synthesis of IL1‐β which may have the potential to cause immune system dysregulation.

Support or Funding Information

Supported by NIH grant U54CA163066.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Calsequestrin, a new modulator of unfolded protein response in skeletal and cardiac muscle
Authors: Wang Qian , Groenendyk Jody , Kor Kaylen , Liu Yingjie , Hiess Florian , Chen S.R. Wayne , Knollmann Björn C. , Michalak Marek
Published: June 2021

Cellular responses to stress are an integral part of muscle physiology and pathology. To deal with stress, cardiac and skeletal muscle cells utilize various mechanisms, engaging intercellular organelles including the endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) [1]. The disruptions of ER functions are often observed in ischemic, hypertrophic and failing hearts [1]. However, the molecular mechanisms governing muscle ER stress regulation in the muscle are still unclear. Disruption of ER/SR hemostasis with physiological and pathological stimuli triggers a coping response, so called unfolded protein response (UPR). One of the UPR signaling pathways involves stress sensor inositol‐requiring enzyme 1α (IRE1α), an integral ER/SR membrane protein kinase and mRNA splicing enzyme. Here, we discovered that calsequestrin, a muscle Ca2+ binding protein localized to junctional SR, interacts with UPR stress sensor IRE1α, and contribute to the modulation of stress responses in the muscle. Both skeletal and cardiac isoforms of calsequestrin bind directly to IRE1α as measured by Microscale Thermophoresis (MST), Surface Plasmon Resonance (SPR), and His‐tag Ni‐NTA‐agarose pull down techniques. Although calsequestrin contains three thioredoxin‐like domains its interaction with IRE1α did not involve cysteine residues in IRE1α. Iodixanol gradient fractionation and immunostaining of skeletal muscle sections or isolated cardiomyocytes showed enrichment of IRE1α protein in junctional SR where it is colocalized with calsequestrin. MST analysis revealed that calsequestrin binding to IRE1α has inhibitory effect on IRE1α by preventing dimerization/oligomerization IRE1α required for X‐box binding protein 1 (XBP1) splicing activity. Consequently, cardiac calsequestrin‐deficient cardiomyocytes have increased activity of IRE1α. We concluded that calsequestrin is a new IRE1α binding partner and the protein plays a role in modulation of stress responses in skeletal and cardiac muscle.

Support or Funding Information

Canada Institutes of Health Research (CIHR)

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 binding interactions of AT hook motif variants
Authors: Dobbins Kaitlyn R. , Buchmueller Karen L.
Published: June 2021

High Mobility Group A (HMGA) proteins are a family of architectural transcription factors that control expression of many genes and their overexpression has been linked to metastasis in numerous cancers. HMGA binds to the minor groove of AT‐rich DNA through three positively‐charged AT hook motifs, each containing the invariant sequence Arg‐Gly‐Arg‐Pro. We endeavor to investigate the effects of changing the amino acids flanking this invariant sequence. Peptides were designed to have varying flanking sequences based on amino acid occurrences in human AT hook proteins. Variations in binding affinities and structural characteristics of these sequences were elucidated using fluorimetry, circular dichroism, and filter binding. Analysis of these variations reveals which amino acids and which amino acid positions in the sequence may be critical to the binding interactions between the AT hook and DNA. These conclusions will inform the development of HMGA‐targeting therapeutics.

Support or Funding Information

Arnold and Mabel Beckman 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.
Mitophagy during Differentiation of Human Embryonic Stem Cells
Authors: KAO LI‐PIN , Wolvetang Ernst
Published: June 2021

Mitochondria are cellular organelles that are critical for energy production. They also serve as signalling hubs, integrating a variety of inputs related to metabolic demand, redox control, and apoptosis. The mass and quality of mitochondria within a cell are determined by a balance between mitochondrial biogenesis and mitochondrial turnover. In human embryonic stem cells (hESCs), mitochondrial quality control is especially critical, as this inner‐cell‐mass derived cells are pluripotent and can propagate indefinitely. Cultured hESCs accumulate mutations in the mitochondrial genome, a fact that may limit the use of hESCs (and their differentiated derivate) in regenerative medicine. Mitophagy is a selective form of autophagy in which a mitochondrion is sequestered within a vesicle, delivered to the lysosome, and degraded. Here we show that the number of mitophagic events increases during early stages of hESC differentiation, as indicated by elevated co‐localization between LC3‐GFP‐labeled autophagosomes and MitoTracker‐labeled mitochondria. Furthermore, we detect increased levels of mitophagy in hESCs treated with agents that perturb mitochondrial integrity (e.g., EtBr, which damages mitochondrial DNA, and CCCP, which depolarizes the mitochondrial membrane). Our data demonstrate for the occurrence of mitophagy within hESCs. Our findings further suggest that mitophagy may selectively remove damaged mitochondria as hESCs differentiate. A more thorough understanding of the molecular mechanisms controlling mitophagy in hESCs may eventually allow for manipulation of this process, a technology that could improve the mitochondrial quality of cultured hESCs and thus facilitate the therapeutic application of these cells in regenerative medicine.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dysregulation of Human Mitochondrial ClpP Protease Activity by Acyldepsipeptides Analogs Leads to Apoptotic Cell Death
Authors: Houry Walid A.
Published: June 2021

Acyldepsipeptides (ADEPs) are potential novel antibiotics. They dysregulate the activity of the highly conserved tetradecameric bacterial ClpP protease leading to bacterial cell death. Here, we identified several ADEP analogs that are potent dysregulators of the human mitochondrial ClpP (HsClpP). These ADEPs interact tightly with HsClpP causing the protease to non‐specifically degrade model substrates. Dysregulation of HsClpP activity by these ADEPs was found to have cytotoxic effects in multiple human cell lines as a result of the activation of the intrinsic, caspase‐dependent apoptosis. ADEP‐HsClpP co‐crystal structure was solved for one of the analogs revealing a highly complementary binding interface formed by two HsClpP neighbouring subunits but, unexpectedly, with HsClpP in the compact conformation. Given that HsClpP is highly expressed in multiple cancers and has important roles in cell metastasis, our findings suggest a novel therapeutic potential for ADEPs in cancer treatment.

Support or Funding Information

This work was funded by Natural Sciences and Engineering Research Council of Canada grant (RGPIN 2014‐05393) to WAH

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Autophagic clearance of proteasomes in yeast requires the conserved sorting nexin Snx4
Authors: Howell Lauren A. , Nemec Antonia A. , Murray Matthew A. , Tomko Robert J.
Published: June 2021

Turnover of the 26S proteasome by autophagy is an evolutionarily conserved process that governs cellular proteolytic capacity and eliminates inactive particles. In most organisms, proteasomes are located in both the nucleus and cytoplasm. However, the specific autophagy routes for nuclear and cytoplasmic proteasomes are unclear. Here, we investigate the spatial control of autophagic proteasome turnover in budding yeast (Saccharomyces cerevisiae). We found that nitrogen starvation‐induced proteasome autophagy is independent of known nucleophagy pathways, but is compromised when nuclear protein export is blocked. Further, via pharmacological tethering of proteasomes to chromatin or the plasma membrane, we provide evidence that nuclear proteasomes at least partially disassemble before autophagic turnover whereas cytoplasmic proteasomes remain largely intact. A targeted screen of autophagy genes identified a requirement for the conserved sorting nexin Snx4 in the autophagic turnover of proteasomes and several other large multisubunit complexes. We demonstrate that Snx4 cooperates with sorting nexins Snx41 and Snx42 to mediate proteasome turnover, and is required for the formation of cytoplasmic proteasome puncta that accumulate when autophagosome formation is blocked. Together, our results support distinct mechanistic paths in the turnover of nuclear versus cytoplasmic proteasomes, and point to a critical role for Snx4 in cytoplasmic agglomeration of proteasomes en route to autophagic destruction.

Support or Funding Information

Florida State University College of Medicine and NIH/NIGMS grant 1R01GM118600

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 Method for Studying Zinc Deficiency in vitro and its Application
Authors: Richardson Christopher Edward , Cunden Lisa Stephanie , Butty Vincent L. , Nolan Elizabeth M. , Lippard Stephen J. , Shoulders Matthew D.
Published: June 2021

Zinc deficiency is correlated with many pathologies, but the molecular connection between disease states and insufficient zinc is still poorly understood. To investigate the connection between zinc deficiency and organismal phenotypes, we developed a technique to specifically diminish zinc levels in mammalian cell culture media without perturbing the concentrations of other metal ions. Growth of cells in such depleted medium evokes some of the transcriptional responses previously noted in organismal models of zinc deficiency, which can be restored upon addition of zinc to the original levels. RNASeq and miRNA data similarly support a zinc‐specific response to the depletion. We also report how the zinc transcriptome remodels in the context of zinc limiting growth. This robust and transportable methodology offers investigators a unique tool for studying zinc deficiency in the context of mammalian cell culture.

Support or Funding Information

The work was supported by the US National Institutes of Health and the Mallinckrodt Faculty Scholar Award.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulating ER Protein Folding Homeostasis By Distinctively Processing mRNAs
Authors: Li Weihan , Okreglak Voytek , Peschek Jirka , Kimmig Philipp , Walter Peter
Published: June 2021

The endoplasmic reticulum (ER) is the major folding compartment for most secretory and plasma membrane proteins in the cell. A conserved signaling pathway, the unfolded protein response, senses and modulates the folding capacity of the ER. To maintain ER protein folding homeostasis under ER stress conditions, the ER membrane embedded sensor, Ire1, initiates two distinct mRNA processing programs through its cytoplasmic kinase/RNase domains. First, in both metazoans and S. cerevisiae, Ire1 catalyzes the unconventional cytoplasmic mRNA splicing of XBP1 (metazoans) or HAC1 (S. cerevisiae)—thereby initiating a transcriptional response that increases the ER folding capacity. Second, in metazoans and S. pombe, Ire1 selectively degrades ER‐localized mRNAs—thereby post‐transcriptionally reducing the ER's protein folding burden through regulated Ire1‐dependent mRNA decay. Thus, Ire1 homologs in S. cerevisiae and S. pombe are specialized to only one of the two functional outputs, while Ire1 in metazoans can perform both. We found that S. cerevisiae and S. pombe Ire1 have distinct RNase specificity, which determine their functional outputs. In addition, we nailed down to three Ire1 residues, which regulate Ire1 RNase specificity by regulating its dimer‐packing. Finally, by applying our new findings, we successfully reconstituted unconventional mRNA splicing in S. pombe cells. Therefore, we engineered S. pombe into a metazoan‐like Ire1 system, where unconventional mRNA splicing and selective mRNA decay co‐exist. Our results provide new insights into a mechanistic understanding of Ire1 function and its interplay with RNA substrates.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DUB activity of Endothelial A20 maintains and repairs endothelial barrier after inflammatory lung injury
Authors: Soni Dheeraj , Wang DongMei , Regmi Sushil C. , Vogel Stephen M. , Tiruppathi Chinnaswamy
Published: June 2021

Vascular endothelial cadherin (VE‐cad) expression at endothelial adherens junctions (AJs) stabilizes endothelial barrier. Inflammatory mediators induced VE‐cad phosphorylation and ubiquitination promotes its degradation; which in turn destabilizes endothelial barrier to trigger vascular inflammatory responses. However, the intrinsic molecular mechanisms involved in maintaining the basal endothelial barrier stability and the repair of endothelial barrier after vascular injury are poorly understood. Here, we generated endothelial cell (EC)‐restricted Tnfaip3 (A20) knockout (A20ΔEC) mice and showed the pivotal role of the deubiquitinase (DUB) function of A20 in regulating the expression of VE‐cad at AJs. A20ΔEC mice failed to survive sub‐lethal dose of LPS challenge. Consistent with this observation, A20ΔEC mice displayed uncontrolled lung vascular leak and persistent PMN sequestration in lungs in response to LPS. In support of these findings, we observed markedly reduced VE‐cad expression in lungs of A20ΔEC mice. Liposome mediated in vivo delivery of WT‐A20 but not DUB‐inactive (C103A‐A20) mutant construct restored VE‐cad expression in lungs, suppressed LPS induced lung vascular leak and sequestration of PMNs in lungs of A20ΔEC mice. Further, A20 knockdown in human lung microvascular ECs resulted increased VE‐cad phosphorylation on Y685 and Y731, and loss VE‐cad expression at AJs. Forced expression of WT‐A20 but not DUB‐inactive mutant in ECs prevented VE‐cad ubiquitination and degradation. Since induced A20 expression promotes resolution of inflammation, we studied the mechanism of A20 induction in response to TLR4 activation in ECs. Here we observed that IRAK‐M‐mediated NF‐κB activation independent of TAK1 is essential for A20 expression in ECs. Importantly, silencing of IRAK‐M in mouse lung microvessels suppressed expression of A20 and VE‐cad and augmented LPS induced lung vascular inflammatory responses. These findings support that targeting endothelial‐A20 expression is a potential therapeutic strategy to restore endothelial barrier integrity in acute lung injury.

Support or Funding Information

National Institute of Health Grants R01 HL‐128539 and R01 GM‐117028

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 Cationic Residue Coordinated to the N1/2O‐Position of FMN in the Nitroreductase Family is Highly Conserved yet not Central to Catalysis
Authors: Musila Jonathan M. , Rokita Steven E.
Published: June 2021

Enzymes of the nitroreductase structural superfamily form an α+β fold and exist as homodimers with active sites formed at the dimer interface. Although these enzymes contain many conserved residues, their flavin chemistry and catalytic activities are wide‐ranging. For instance, nitroreductase (NfsB) and flavin mononucleotide (FMN) reductases exclusively promote reaction by concurrent transfer of two electrons while iodotyrosine dehalogenase (IYD) and 5,6‐dimethylbenzimidazole synthase (BluB) primarily promote reaction by two single electron transfers. A cationic residue (Arg/Lys) coordinates the N1/2O of the bound FMN throughout the superfamily regardless of their catalytic specificity. To test the necessity of such a highly conserved residue, the candidate residue in IYD (Arg38) was alternatively mutated to a neutral (Gln) and anionic (Glu) derivative. Additionally, the catalytic necessity of an Arg over a Lys was evaluated. The affinity of the R38K and R38Q IYD mutants for iodotyrosine (I‐Tyr) decreased by 11‐ and 46‐fold respectively, compared to that of the wild‐type IYD. In addition, the dehalogenation reaction promoted by R38K and R38Q IYD were suppressed by ~10 and 20‐fold. The most extreme substitution of R38E exhibited no detectable dehalogenation activity but did not prevent FMN from binding to the active site. The R38E IYD also produced a 78‐fold decrease in I‐Tyr affinity.

The Arg38 mutations did not, however, alter the FMN chemistry of IYD suggesting that the Arg/Lys at the N1/2O‐position of FMN is not crucial in regulating the one versus two electron transfer process. All variants had comparable FMNox/FMNhq midpoint potentials relative to that of wild‐type IYD. The one electron chemistry of IYD is only expressed in the presence of its substrate or an inert substrate mimic such as fluorotyrosine (F‐Tyr). In the presence of F‐Tyr, the R38Q and R38E variants exhibited an increase of ~50 mV for the midpoint potential of the FMNox/FMNsq couple. In addition, the R38Q and R38E variants registered an increase in the FMNsq/FMNhq midpoint potential of ~50 mV and ~90 mV respectively. Although the R38K IYD had a FMNox/FMNsq midpoint potential comparable to that of the native enzyme, its FMNsq/FMNhq mid‐point potential also increased by ~50 mV. We anticipate similar results with the other model nitroreductases (NfsB and BluB) in this investigation.

Support or Funding Information

JHU Provost's Postdoctoral Fellowship and Discovery Award

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparing E. coli Expression and Enzyme Kinetics of Wildtype and Codon Optimized 2‐(2′‐hydroxyphenyl)benzenesulfinate desulfinase (DszB) from Nocardia asteroides A3H1 and Rhodococcus erythropolis IGTS8
Authors: St.George Marissa Louise , Idrizi Keid , Watkins Linette M.
Published: June 2021

Dibenzothiophene (DBT) and its derivatives comprise up to 60% of the organosulfur contamination of crude oil. The enzyme 2‐(2′‐hydroxyphenyl)benzenesulfinate desulfinase (DszB) catalyzes the carbon‐sulfur bond cleavage in the final, and rate‐limiting step in the biodesulfurization of DBT. The wildtype dszb genes from Nocardia asteroides A3H1 (A3H1) and Rhodococcus erythropolis IGTS8 (IGTS8) were expressed with and without chaperone proteins under different induction conditions. Yield of active enzyme was optimal when co‐expressed in E. coli in the presence of GroEL and GroES. Codon optimized dszb genes were synthesized, optimizing E. coli codon usage and minimizing GC content. The codon optimized dszb genes from A3H1 and IGTS8 were expressed with and without chaperone proteins under different induction conditions. GroEL and GroES were both still required for optimal enzyme activity. Protein aggregation was observed using whole cell FT‐IR spectroscopy in all cases, but was significantly larger in induced cells in the absence of chaperones.

Support or Funding Information

NSF CHE 1461175

James Madison University Department of Chemistry and Biochemistry

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Natural compounds as probes to dissect lysosome homeostasis
Authors: Li Yang , Hao Xiaojiang , Yang Chonglin
Published: June 2021

Lysosomes are acidic single‐membrane organelles that serve as the major sites for degradation of cargoes delivered from endocytosis, phagocytosis and autophagy. Recent studies have established lysosomes as important hubs that couple metabolic signaling with cellular clearance. Dysfunction of lysosomes contributes to many human disorders such as lysosome storage diseases (LSDs) and neurodegenerative diseases. To better understand the mechanisms underlying lysosome homeostasis, we screened natural small‐molecule compounds that induce lysosome biogenesis or inhibit lysosome functions. A group of diterpenes, which are activators of protein kinase C (PKC), were found to induce lysosome biogenesis by activating the TFEB transcription factor and inactivating the ZKSCAN3 transcriptional repressor in an mTOR‐independent manner1. In contrast, several tetranortriterpenoids, represented by HLSP, appeared to inhibit the acidification of lysosomes. We found that HLSP also affects intracellular trafficking and autophagy. We are now in the process of investigating the mechanisms of HLSP action by identifying its protein target that is required for generation and maintenance of lysosomal acidity.

Support or Funding Information

This research was supported by grant 21432010 (to X.Hao and C.Yang) from the National Science Foundation of China.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Design and Synthesis of a Triazole‐based Small Molecule Library for the Inhibition of Bacterial Quorum Sensing
Authors: Kruszynski Catherine E. , Hinspeter Kallie , Danowitz Amy M.
Published: June 2021

With the rise of antibiotic resistance, finding novel methods of targeting bacteria and treating disease is in high demand. One target system of interest is the bacterial quorum sensing system. This process involves bacteria communicating using small signaling molecules. This communication allows bacteria to engage in group behaviors such as biofilm formation and release of infective agents. It has been demonstrated that synthetic small molecules can be used to inhibit such behaviors. Although some success has been had, the design of more diverse libraries could lead to more efficient modulators, easier synthetic routes, and increased understanding of the quorum sensing pathway. The research presented here focuses on the design and synthesis of a library of adamantyl‐derived triazoles. The triazole scaffold was chosen because of its ease of synthesis, and a wide selection of terminal and di‐substituted alkynes were chosen based on similarities to natural quorum sensing molecules. Some functionalities include, a five membered ring with heteroatoms and carbon chains of varying lengths. Upon synthesis and purification, these molecules are assayed for their ability to inhibit quorum sensing in Vibrio fischeri. Vibrio fischeri is used because its quorum sensing system is similar to other Gram‐negative bacteria that are known to cause human infections (i.e. Pseudomonas aeruginosa), but it is non‐pathogenic. Additionally, this species' bioluminescence is controlled by a quorum‐dependent pathway, which allows for easy detection of quorum sensing inhibition via quantification of light production. To date, the molecules that have been tested have not been found to inhibit bacterial quorum sensing, however, the library continues to grow with different functionalities. Herein, we will describe the design and synthesis of a growing triazole‐based library as well as data regarding the ability of these molecules to inhibit quorum sensing in Vibrio fischeri.

Support or Funding Information

CEK received partial funding from the Pennsylvania Academy of Sciences.

Mercyhurst University provided the remaining funding.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigations of Essential Oils as Quorum Sensing Inhibitors of Vibrio fischeri
Authors: Hinspeter Kallie , Danowitz Amy M.
Published: June 2021

The misuse of traditional antibiotics has gradually caused a decrease in their efficacy. As bacterial resistance continues to spread, the need for the development of novel methodologies to prevent bacterial development has increased. One area of focus in this field is the inhibition of quorum sensing, a process by which bacteria are able to communicate and collectively regulate gene expression in a cell‐population density dependent manner through small molecules called autoinducers. By blocking cellular receptors which bind autoinducers, bacterial communication can be impeded. This can cause a decrease in community behavior including biofilm formation, production of virulence factors, and bioluminescence. Unlike traditional antibiotics, inhibiting quorum sensing obstructs virulence without preventing bacterial growth and is therefore thought to reduce the evolutionary drive for resistance development. Natural plant products, such as phytochemicals including phenolics, flavonoids, quinones, and alkaloids, have been shown to inhibit virulence factor production, quorum sensing, and microbial growth. This research initially aimed to examine the quorum sensing inhibition properties of select essential oils on Vibrio fischeri, a bioluminescent bacteria, through photometric assays. As the bioluminescent properties of Vibrio fischeri function through a quorum‐dependent pathway, the examination of this species allows for the easy detection of quorum sensing inhibition through the quantification of light production. Essential oils tested include caraway (Carum and Carvi oils), cistus or labdanum (Cistus ladaniferus), elemi (Canarium luzonicum), fir needle (Abies sibirica), and pine (Pinus sylvestris). Ongoing experimental results suggest that the essential oils originally selected for this research display antibacterial properties over quorum sensing inhibition. Due to this, future directions will likely move towards an examination of the antibiotic properties of these oils in Escherichia coli, a very common Gram‐negative pathogen that can be harmless in certain strains.

Support or Funding Information

Funding for this project was provided by Mercyhurst 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.
Accessing new chemical diversity from ancient non‐actinobacterial strains
Authors: Terrell Stephanie M. , Wu Yang , Klein Joshua G. , Charkoudian Louise K.
Published: June 2021

Our work focuses on the discovery of new biosynthetic routes to chemical diversity through the characterization of unexplored polyketide synthase (PKS) gene clusters and enzymes from ancient and diverse phyla. In the first part of this work, two ancient non‐actinobacterial organisms were cultured, and their secondary metabolites extracted, fractionated, and characterized by liquid chromatography mass spectrometry (LC‐MS) in the search for novel molecules and biosynthetic pathways. These data are currently being compared to existing molecular networks in order to identify novel molecules. In a second part of this work, ancient non‐actinobacterial strains are being evaluated as a source of PKS enzymes that can be expressed in tractable heterologous hosts to enable in vitrocharacterization of poorly‐understood PKSs. We have successfully expressed a ketosynthase‐chain length factor (KS‐CLF) heterodimer pair, which is essential in the manufacturing of polyaromatic polyketides, in Escherichia coli, and are currently evaluating its activity in vitro. Results from these studies are expected to lead to the identification of new chemical diversity encoded by nature, as well as novel tools to enable the biosynthetic engineering of hybrid PKSs.

Support or Funding Information

We would like to acknowledge an NSF CAREER Award #R15GM120704 to Louise K. Charkoudian as a source of funding for this project.

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