bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transcriptome Analysis of Conditional Knockout Mice
Authors: Biefeld Chelsea , Sztroin Kelcie , Zhu Yi , Yu Gang , Dries Daniel 
Published: November 2023

Gamme‐secretase is an intramembrane protein that cleaves type I transmembrane proteins. It is known to be involved in Alzheimer's disease and neurodevelopment. Previous studies have investigated the role of gamma‐secretase role in neurons however, it has seldom been considered in glial cells. Here, a conditional knockout mouse was made in which gamma‐secretase was rendered nonfunctional in oligodendrocytes. These mice displayed schizophrenic‐ and compulsive‐like phenotypes. In order to identify genes and pathways that elicit these phenotypes, RNA‐Seq was used to perform transcriptome analysis searching for differentially expressed genes in the experimental mice. Gene ontology programs were used to evaluate the genes and identify biological processes that may play a role in the behavioral aspects of these mice.Support or Funding InformationWe would like to thank the Howard Hughes Medical Institute, the National Institutes of Health (Grant R01 AG029547), and the International Healthspan Institute for providing funding.
The molecular basis for cross‐species prion transmission
Authors: Kurt Timothy , Jiang Lin , Alderson Nazilla , Liu Jun , Eisenberg David , Sigurdson Christina 
Published: November 2023

BackgroundA key event in the pathogenesis of prion diseases is the conversion of the cellular prion protein, PrPC, into a misfolded, self‐templating conformer called PrPSc. The mechanism by which PrPSc templates the misfolding of PrPC is unknown. We previously demonstrated that the amino acid sequence within key segments of PrPC has a profound impact on conversion by PrPSc. For instance, two critical residues, E168 and S170, impede cross‐seeding of human PrPC by chronic wasting disease (CWD) prions from elk and deer1. Interestingly, bank voles, a small European rodent, are highly susceptible to prions from diverse species including humans, elk and mice, and have been referred to as the “universal acceptor for prions”2. The molecular basis for the remarkable susceptibility of bank voles to prion infection is unknown..HypothesisWe hypothesize that the bank vole PrPC sequence is susceptible to prion cross‐seeding due to key amino acids that are within key adhesive segments of the protein that are critical for prion aggregation. Further, we hypothesize that substituting the key bank vole residues into the PrPC from other species will enable conversion of PrPC from any species.MethodsI used an in vitro prion conversion assay called cell‐lysate protein misfolding cyclic amplification (PMCA) to probe the effects of amino acid substitutions on the conversion of bank vole PrPC. PrPSc from elk, mice and sheep was used to seed conversion of PrPC. Data were measured by semi‐quantitative western blotting and analyzed by ANOVA and t‐test.Results and ConclusionsWe found that asparagine and glutamine amino acids are enriched among the key bank vole residues, suggesting that asparagine ladders and polar zippers may underlie the high susceptibility of bank vole PrPC to prion conversion. These data provide important insight into the structure of infectious prions, as the key residues must comprise an interface for interaction with PrPSc. Interestingly, we also found evidence suggesting that an interaction between residues in the N‐terminal disordered domain and the globular domain of PrPC impacts prion conversion. This finding has not been previously reported and indicates that the N‐terminal domain, which is not considered part of the β–sheet core of the misfolded prion, plays an important role in prion conversion. In addition, we have confirmed that heterologous amino acids residues may promote prion cross‐seeding, an important finding that is relevant to understanding cross‐species prion transmission and zoonotic disease risk.Support or Funding InformationThis work was supported by NIH grant NS069566 and NS076896 (CJS) and 1 K01 0D019919‐01 (TDK).
Effect of rLosac (Lonomia obliqua stuart‐factor activator) on skin wound healing
Authors: de Andrade Sonia Aparecida , Sato Ana Claudia , Alvarez‐Flores Miryam Paola , Bosch Rosemary , Chudzinski‐Tavassi Ana Marisa 
Published: November 2023

Belonging to the hemolin family, Losac is a factor X activator, found on bristles of the Lonomia obliqua. Its features cytoprotective activity and modulates the expression of extracellular matrix molecules in a fibroblast culture. To study the effect of rLosac on skin wound healing in rats. Approval # 1254/14 by Comissão de Ética no Uso de Animais do Instituto Butantan. Twenty Wistar (Rattus norvergicus albinus) male rats, aged 6–8 weeks, weight 120 – 150 g obtained from Biotério Central do Instituto Butantan. Rats were submitted to oral preemptive analgesia 200 mg/kg paracetamol and kept on post‐surgery procedures. The animals were anesthetized by intraperitoneal injection of 75 mg/kg ketamine and 10 mg/kg xylazine. Four full‐thickness excisional 4 sq mm punches were created through the skin. The wounds of the animals were treated topically with 63.80 μg/mL rLosac (L1), 127.60 μg/mL of rLosac (L2) or saline solution (SS). Zero, 3, 7, 14 and 21 days after skin injury, the wounds were measured using a digital caliper rule and the rats euthanized with excess anesthetic. The areas corresponding to the lesion were removed with scalpel and analyzed. The three independent treatment groups were analyzed through the Kruskall‐Wallis test on days 0, 3, 7, 14 and 21. Regarding the initial area (12.56 sq mm), after three days of treatment the wound was reduced by 63.5 % on L1 group, 75.0 % on L2, and 51.25 % on SS. After seven days, the initial area was reduced by 72.76 % on SS; 87.5 % on L1 and 94.75% on L2. After 14 and 21 days of treatment, just scars were observed. The rLosac accelerates wound healing. The gelatinolytic activity from MMP‐2 increased on days zero, 3 and 14 on the wound treated with rLosac. However, on day 7 there was a decreased activity. MMP‐2 degrades basement membrane collagens and demots denatured structural collagens. Proteolytic degradation of the extracellular matrix is an essential feature of repair and remodeling during wound healing. On day zero, L2 featured two more intense spots, possibly corresponding to the α1 and α2 chains of the type I collagen when compared to control. There is also an approximately 210 KDa spot, probably corresponding to the β chain. On day three the spots remained more intense in L1 and L2 when compared to SS and to day zero. On days 7, 14 and 21 there was a significant increase on the intensity of the spots, possibly corresponding to the α1 and from the α2 type I collagen, with the α1 chain more intense when compared to the α2 chain. These initial results have shown that the healing time of the wounds treated with rLosac is significantly smaller when compared to the control, which suggests the potential benefit of this protein on wound healing.Support or Funding InformationSupport by FAPESP
Structural determinants of human tryptophan 2,3‐dioxygenase (TDO) protein degradation
Authors: Kim Sungmi , Karkashon Shay , Yeh Syun‐Ru , Correia Maria Almira 
Published: November 2023

The hepatic hemoprotein tryptophan 2,3‐dioxygenase (TDO) catalyzes the irreversible oxidation of L‐tryptophan (Trp) to N‐formyl‐L‐kynurenine. It critically regulates the flux of Trp through the kynurenine and serotonin pathways, thereby controlling systemic Trp levels. TDO has been implicated in various diseases, including neurodegenerative diseases and cancers, and thus is an important target for drug discovery. Despite its importance, very little is known about the regulation of human TDO (hTDO) protein turnover required for its cellular homeostasis. Herein we report that hTDO turnover occurs via ubiquitin‐linked proteasomal degradation (UPD) pathway, and we define the structural features critical therein. Trp, the preferred substrate is known to “induce” TDO enzyme via stabilization. Structural analyses reveal that Trp binds hTDO at both the active site and an exo‐site that also binds aMethylTrp (aMeTrp). 35S‐pulse‐chase analyses in HepG2 cells revealed that exo‐site aMeTrp‐binding prolonged the half‐life of parent wild‐type (WT) hTDO 2‐fold. Site‐directed mutagenesis of exo‐site residues considerably accelerated hTDO degradation, despite aMeTrp presence. By contrast, deletion of hTDO N‐ and/or C‐terminus (ΔN, ΔC, ΔN/ΔC) greatly stabilized the protein in the order ΔN/ΔC>ΔC>ΔN, consistent with a processive proteolytic process. The extreme hTDO C‐terminus is rich in acidic (Asp/Glu) and phosphorylatable (Ser) residues. Relative to the WT, mutation of these Asp/Glu‐residues to Gly stabilized hTDO, whereas that of Ser to Gly had no effect. When the exo‐site mutation was incorporated into the hTDOΔN/ΔC mutant, its stability was lost, being degraded just as rapidly as the exo‐site hTDO‐mutant. Collectively these findings reveal that although the exo‐site along with the N‐ and C‐termini are important structural features in hTDO turnover, the structural integrity of the exo‐site Trp‐binding is a critical determinant of its UPD.Support or Funding InformationNIH grants GM44037, DK26506, and GM086482.
p97 is Critical for Cardiac Proteostasis and Dysregulation of p97 Activity Causes Cardiomyopathy
Authors: Brody Matthew J , Karch Jason , Molkentin Jeffery D 
Published: November 2023

Endoplasmic reticulum (ER)‐associated degradation (ERAD) is a protein quality control process whereby proteins that cannot be properly folded in the ER are retrotranslocated to the cytosol for degradation by the ubiquitin proteasome system. p97 is a AAA‐ATPase that forms a ring‐shaped hexameric structure on the cytosolic side of the ER membrane and provides the energy to retrotranslocate misfolded substrates from the ER for ultimate ubiquitin‐dependent degradation. Mutations in p97 are known to cause protein aggregation‐based diseases including pathologies of the nervous system, skeletal muscle, and heart. However, the role of p97 in cardiomyocytes has not been examined. Here, we generated cardiac‐specific transgenic mice overexpressing wildtype p97 or a p97K524A mutant with deficient ATPase activity. Transgenic mice overexpressing wildtype p97 exhibit normal cardiac structure and function while mutant p97 overexpressing mice develop age‐dependent cardiomyopathy at baseline and greatly exacerbated cardiomyopathy in response to pressure overload. Mechanistically, both wiltype and mutant p97 overexpressing hearts upregulate the protein levels of Sel1l, an ERAD adaptor protein, while p97K524A overexpressing hearts additionally upregulate the ERAD complex components OS‐9, Herp, and the ER‐resident prolyl isomerase cyclophilin B. Importantly, hearts overexpressing p97K524A but not wildtype p97 have elevated levels of ubiquitinated proteins, indicating that p97 activity is required for efficient clearance of proteins targeted for proteasomal degradation. Moreover, overexpression of wildtype but not mutant p97 in cardiomyocytes enhanced ERAD activity and protected from ER stress induced cell death. These studies identify a critical role for p97 in cardiac proteostasis and demonstrate that dysregulation of p97 activity causes cardiomyopathy.Support or Funding InformationThis work was supported by a National Research Service Award (F32HL124698) from the National Institutes of Health to M.J.B. and grants from the Howard Hughes Medical Institute and National Institutes of Health (R01HL105924) to J.D.M.
Skeletal Muscle Wasting and Inflammatory Regulation by Ovarian Function and IL‐6 in Tumor Bearing Mice
Authors: Reszczynski Olivia , Saum Alison , Hetzler Kimbell , Carson James 
Published: November 2023

Cancer cachexia is a progressive wasting condition involving the loss of muscle and fat, as well as biochemical abnormalities due to chronic underlying disease such as cancer. IL‐6, a pro‐inflammatory cytokine, has been shown to be a major driver of cancer cachexia in several mouse models, including the male ApcMin/+ (Min) mouse, and humans. Studies regarding cachexia and IL‐6 have largely been conducted in male mice; however, our lab has noted sex differences in cachexia development and IL‐6 response using the ApcMin/+ mouse. Inflammation related to IL‐6 signaling is associated with myofiber wasting in the male ApcMin/+ mouse, but it has been unexplored in the female. Though it has been seen that hypogonadism is a factor in cachexia in males, research has not been conducted to determine the effect of hypogonadism in the female. Given estrogen's known anti‐inflammatory effects, it is possible that ovarian endocrine function plays a role in the differential female IL‐6 response. The purpose of this study is to determine the effect of ovarian function and the role of IL‐6 in the muscle wasting seen during progression of cachexia. The hypothesis was that ovarian function loss (OVX) with IL‐6 overexpression would cause an increase in muscle inflammation, leading to increased myofiber wasting and decreased myofiber area. ApcMin/+ mice were divided among four groups: control (n=13), sham+IL‐6 overexpression (n=10), OVX+vector (n=10), OVX+IL‐6 (n=8). Tibialis anterior (TA) muscles were sectioned and mounted on slides. H&E staining was performed to determine myofiber cross‐sectional area (CSA). A greater proportion of small (<1500 μm), and smaller proportion of large (>2000μm) fibers were found in sham+IL‐6 mice vs. control mice (p<0.001), as well as in OVX+IL‐6 versus OVX+vector mice (p<0.001), indicating that IL‐6 overexpression induces a shift towards smaller fibers independent of ovarian function. Membrane receptor gp130, required for IL‐6 intracellular signaling, protein expression increased with IL‐6 overexpression and attenuated by OVX. OVX also decreased STAT3 phosphorylation, an intracellular mediator of IL‐6 signaling. Increased inflammation related to IL‐6 signaling is associated with muscle wasting in the female ApcMin/+ mouse and is attenuated by loss of ovarian function.Support or Funding InformationThe authors would like to acknowledge Justin Hardee, Dennis Fix, Song Gao, and Brandon VanderVeen for technical assistance. This research was funded by NIH grant NCI R01CA121249‐01.
Understanding The Lipid Mediated Oligomerisation In Membrane Proteins
Authors: Gupta Kallol , Robinson Carol 
Published: November 2023

A quarter of the human proteome is composed of membrane proteins (MP). Nevertheless, less than 2% of known protein structures are of MPs and less than a quarter of those contain any modelled lipids. To date, no multimeric MP structures exist in the protein structure database with lipids at the protein‐protein interface. This highlights the limitation of crystallography in determining the identity of lipid molecules, owing to their conformational flexibility and high mobility. The structural and functional effects resulting from the interactions between MPs and their surrounding lipids have been the subject of constant debate. A major facet of these discussions is the role of lipids in regulating the oligomeric states of MP assemblies. To address this an extensive bioinformatics analysis of all MP structures led to a set of model systems with weak interfaces, potentially suggesting lipid mediation. Exploiting the ability of native‐mass spectrometry (nMS) to simultaneously observe the binding of lipids to MP and their impact on oligomeric states we have developed for the first time a model for lipid‐induced oligomerisation of a MP in atomistic detail and set up a general platform to study this phenomenon in other MPs. nMS analysis of these MPs revealed a defined mass addition of lipids to the oligomers. Among the target systems, for the bacterial amine transporter LeuT, it was observed that selective removal of specific lipids leads to complete de‐oligomerisation, establishing the essential role of lipids towards maintaining the dimer. For the bacterial semi‐SWEET, a homolog of eukaryotic sugar transporters, equilibrium between the monomer and the functionally active dimeric state was observed. Added lipids were found to specifically bind to and stabilize the active state, elucidating a mechanism through which an organism may select out an active oligomeric state of a membrane protein by modulating the interacting lipids in the membrane. Through an integrative lipid‐analysis, using a novel high‐energy platform that allows MS/MS analysis of MPs, we identified bound phospholipids and cardiolipins. Analysis of delipidated proteins, as well as mutants that lack dimeric interactions and the probable lipid binding sites, proposed through MD simulation, coupled with integrated lipid analysis and live cell imaging, an atomistic model was revealed of how lipids can act as plugs to bridge weakly bound oligomers. The approach, relying on the unique ability of MS to determine the oligomeric states of MPs, with precise identification of bound endogenous lipids, provides a platform that will further the understanding of how lipids can modulate the functional states of oligomeric membrane proteins and trigger specific downstream cellular pathways.Support or Funding InformationKG is a fellow of the 1851 Royal Commission and a Junior Research Fellow at the St Catherine's College, University of Oxford
Using a Toxicogenomic Approach to Understand How Silver Poisons the Bacterial Cell
Authors: Lemire Joe , Chatfield‐Reed Kate , Chua Gordon , Turner Raymond J. 
Published: November 2023

IntroductionThe emergence of the post‐antibiotic era has left us desperate for alternative strategies to combat infectious disease. Historically, silver has been used to prevent and control microbial infections. Silver is now seeing widespread use in a variety of medical devices including bandages, catheters, and endotracheal tubes; as well as numerous household items. Yet, we still don't mechanistically understand how silver poisons the microbial cell.ObjectivesRecently, our research group demonstrated that the higher oxidation states of silver have an enhanced capacity to eradicate laboratory and antibiotic‐resistant strains of bacteria – both planktonic bacteria as well as biofilms. Our current research objective is to understand the mechanistic details of how silver exerts its toxicity on the bacterial cell.MethodologyTo address the mechanisms of how silver kills bacterial cells, we used a high‐throughput, toxicogenomic approach ‐ screening a knockout collection of E.coli K12 (the Keio collection) for Ag‐sensitive/resistant mutants. Following our toxicogenomic screen, markerless deletion mutants were generated from statistically significant hits using the Red Recombinase system. The markerless genetic mutants were then subjected to phenotypic and biochemical assays to verify their resistance or sensitivity profile to silver treatment.ResultsAlthough silver is thought to exert its toxicity by disrupting thiol metabolism and cellular redox‐status, our results demonstrate that silver poisons E.coli through a variety of previously unestablished mechanisms. These include interfering with genes involved in maintaining cell wall integrity, toxin/antitoxin systems, nutrient catabolism, and quorum sensing.ConclusionHere we demonstrate how a toxicogenomic approach was used to determine the mechanistic manner by which silver poisons the bacterial cell. To that end, the robust nature of this methodology can be used to screen other metals, toxins, and antimicrobials to determine their mechanisms of action. Mechanisms that are desperately needed to develop novel antimicrobial strategies.Support or Funding InformationThis work has been supported by the Natural Science and Engineering Council of Canada. J.L. is supported by a Banting Postdoctoral Fellowship and an Alberta Innovates Health Solutions Postdoctoral Fellowship Award.
Absorption Mechanisms of Poly I:C RNA Onto Zinc Oxide Nanoparticles: Maximizing The Payload
Authors: Ramani Meghana , DeLong Robert K 
Published: November 2023

Development of stable RNA‐nanoparticle complexes will advance RNA therapeutics. Given the complexity of bionano interactions, it is crucial to develop efficient RNA loading techniques, to optimize RNA payload. Here we investigated the loading of anti‐cancer RNA (Poly I:C) using two different sizes of ZnO (14, 100 nm) and three different loading techniques; 1) Centrifugation, 2) Incubation 3) Reannealing. The amount of RNA binding per mg nanoparticle was quantitated using the absorption of RNA at 260 nm by UV‐visible absorption spectroscopy. In order to evaluate the number of binding sites and the binding kinetics, the samples were characterized by fluorescence spectroscopy using the modified Stern‐Volmer equation. In general, the RNA payload per mg nanoparticle was observed to be higher for 14 nm nanoparticles than the 100 nm due to larger surface area. Also, reannealing was most efficient with maximum payload of 20μg per mg of the 14 nm nanoparticles. Reannealing enhances the binding constant (kb = 6.8×103 g−1L) as well as the number of binding sites (n = 5) available on the surface of ZnO for one RNA. Taken together the data suggest three different mechanisms of RNA interaction with nanoparticle: physisorption, aggregation, and chemisorption. TEM analysis confirms an RNA corona surrounding the nanoparticle. This is the first example we are aware of an RNA corona surrounding an un‐functionalized inorganic non‐polymeric nanoparticle. Based on our spectroscopy analysis comparing these three techniques, reannealing poly I:C to 14 nm ZnO nanoparticle, demonstrates the highest payload and suggests a possible process for designing the optimal delivery system.Support or Funding InformationThis work was supported by NCI 7R15CA139390‐03
Analysis of Myogenic Markers in Duchenne's Muscular Dystrophy Cell Models
Authors: Harnois Melissa , Pincus Mark , Powers Elaine , Badarinarayana Vasu 
Published: November 2023

The goal of the project was to compare the in vitro differentiation potential of Duchenne's Muscular Dystrophy (DMD) and non‐disease patient derived myoblasts. The differentiation of myoblasts into myotubes as well as the maturity of myotubes was assessed by morphology, immunohistochemistry (IHC) analysis of myotubes markers as well as transcript profiles of myogenesis markers. Human myoblasts derived from three non‐disease and three DMD patients were evaluated in multiple time‐course studies. Morphological evaluation as well as IHC analysis indicated that the DMD patient derived myoblasts have diminished capacity to differentiate and form mature myotubes. Gene expression profiling also revealed significantly reduced basal transcript levels of myogenic markers in DMD patient derived cells and impaired induction of these transcripts during differentiation.Support or Funding InformationSanofiTucson Innovation Center, Oro Valley, AZ