bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Search for Novel Splicing Factors: Mapping Mutations in a Temperature‐Sensitive Schizosaccharomyces pombe Library
Authors: Williams Jelani Akil , Dwyer Zachary , Fair Benjamin , Larson Amy , Pleiss Jeffrey A. , Inada Maki 
Published: April 2020

Gene expression, starting with the transcription of DNA to messenger RNA (mRNA) and the subsequent translation of mRNA transcripts to form proteins is a vital process for all living cells. Newly transcribed RNAs must be processed before they can serve as templates for protein synthesis. RNA splicing is a key step in RNA processing. Defective splicing disrupts the production of functional proteins and is linked to debilitating human diseases such as cancer, frontotemporal dementia, and Duchenne muscular dystrophy. However, the full set of factors involved in the splicing pathway remain unknown. Hence, uncovering the complete set of splicing factors is critical for the development of new therapeutic interventions.A recent sequencing‐based genetic screen
1,
2 of a temperature‐sensitive (ts) Schizosaccharomyces pombe library
3 identified a subset of mutants that displayed global splicing defects, which may highlight essential genes with potentially novel implications in splicing. The identities of the specific mutations for each of the strains in this ts library (N = ~2300) have never been previously mapped. Therefore, we aim to map the mutations in this library and quantify their impacts on RNA splicing, allowing us to identify the genes detected in the recent screen and possibly reveal additional novel splicing factors and functional connections. Here we use Tn5 tagmentation for genome fragmentation and Gibson assembly for the high‐throughput synthesis of a wild‐type (WT) cloning library, which will then be transformed into each mutant strain to identify the mutant alleles via complementation testing. Once the alleles have been identified, we will characterize their global splicing deficiencies by sequencing methods to measure the ratio of unspliced to spliced RNA transcripts for all introns. Our efforts will annotate the ts mutant library for general use and contribute to the knowledge of the elements involved in RNA splicing.
A novel drug inhibits exosome content loading and secretion in ovarian cancer cells
Authors: Vaughn Kiera , Lausen James , Bruns Matthew , Benbrook Doris , Hannafon Bethany 
Published: April 2020

Ovarian cancer is the 5th leading cause of cancer deaths in women due to late stage diagnosis and limited treatment options. Exosomes are nanometer‐sized vesicles produced in multi‐vesicular bodies (MVBs) and released into the extracellular environment. Exosomes play a role in the progression of ovarian cancer by encapsulating and transferring DNA, RNA (including microRNAs), proteins, and lipids between cells and modifying the tumor microenvironment. Data from our laboratory shows that a novel drug binds to proteins involved in exosome secretion, transport, and content loading, including microRNA binding proteins. The goal of this research is to determine whether this novel drug, called tumor exosome inhibitor (TEXi), can prevent secretion and/or content loading of ovarian cancer exosomes. Using an exosome secretion assay (CD63‐nanoluciferase), nanoparticle tracking analysis, and western blot we found that TEXi inhibits exosome secretion in ovarian cancer cells. Ovarian cancer cells expressing CD63 tagged with GFP allowed for visualization of exosome production and co‐staining for lysosomes and mitochondria allowed for the visualization of the relationship between exosome production in MVBs and these other organelles. Treatment with TEXi followed by live and fixed‐cell imaging demonstrated that TEXi inhibits the secretion by promoting fusion of MVBs with the lysosome. Exosome microRNA secretion, measured by qRT‐PCR, was also reduced by TEXi treatment. In conclusion, TEXi can block exosome and microRNA release by promoting MVB lysosomal degradation. This study has identified a potential new strategy for the treatment of ovarian cancer by blocking the effect of exosomes on the tumor microenvironment.Support or Funding InformationNative American Research Center for Health (NARCH), American Society of Investigative Pathology‐Summer Research Opportunity Program in Pathology (ASIP‐SROPP) and Oklahoma Center for the Advancement of Science and Technology (OCAST) HR17‐052.
Activity of NADH‐ and FADH2‐linked mitochondrial respiration are significantly decreased in two mice models of Parkinson’s disease
Authors: Soliz Jorge , Picón Marina Lorente , Doucet-Beaupré Hélène Hélène , Lévesque Martin , Laouafa Sofien 
Published: April 2020

Parkinson’s disease (PD) is a complex neurodegenerative disorder, which etiology is still largely unknown. Overwhelming evidence indicates that mitochondrial dysfunction is a central factor in PD pathophysiology. Here we tested the hypothesis that the activity of complex I and II (NADH‐and FADH2‐linked mitochondrial respiration, respectively) in the substantia nigra are significantly decreased in PD. To test this hypothesis, we used two different mice models of PD: Mice that received a single unilateral intrastriatal injection of 1) 6‐OHDA (9μg ‐ a selective catecholaminergic neurotoxin), or 2) A53T‐α‐synuclein (5.62E12 GC/ml – a molecule that attenuates synaptic vesicle recycling and neurotransmitter release). Control groups were injected with vehicle (fluorescent protein). Mitochondrial oxygen consumption was evaluated by using our oxygraph‐2K system (O2k‐OROBOROS), in 2 mg weight of tissue permeabilized with saponin. Measurements were performed one week after treatment with 6‐OHDA, and five weeks after treatment with A53T‐α‐synuclein. Our preliminary results show that, compared to control animals, substrates of complex I (Pyruvate/Glutamate/Malate), in the presence of ADP (state 3 complex I), induced a reduced oxygen consumption in mice treated with 6‐OHDA,(50.65 ± 2 vs 78 ±3 pmol O2/s/mg tissue, p<0.001). Inline, compared to control animals, oxygen consumption in state 3 complex I, was significantly reduced in mice treated with α‐synuclein (31.05 ± 5 vs 43.56 ± 2 pmol O2/s/mg tissue, p<0.05). Moreover, oxygen consumption in the presence of substrates of the mitochondrial complex II (succinate – state 3‐complex II), was also significantly reduced after treatment with α‐synuclein (30.8 ± 4.5 vs 46.5 ± 5 pmol O2/s/mg tissue, p<0.05). No differences of oxygen consumption were found in the presence of PMG, but the absence of ADP (state 4 – mitochondrial membrane leak) with none of the treatments. We concluded that 6‐OHDA and α‐synuclein, two reliable models of PD, induce mitochondrial dysfunction by decreasing the activity of mitochondrial complexes I and II. Our results contribute to a better understanding of the mitochondrial dysfunction induced by PD, and open the avenue for alternative treatments aiming to avoid the degeneration of dopaminergic neurons in the substantia nigra.Support or Funding InformationJorge Soliz is supported by the “Fonds de recherche du Quebec‐Santé” (FRQ‐S; FQ121919)
Enhanced expression of the full‐length seven transmembrane mu opioid receptor by MOR‐1G, a truncated six transmembrane splice variants through heterodimerization
Authors: Zhang Tiffany , Xu Jin , Pan Ying-Xian 
Published: April 2020

The mu opioid receptor gene, OPRM1, undergoes extensive alternative splicing, generating an array of splice variants. One type of the splice variants are truncated variants that have six transmembrane (TM) domains lacking the first TM. Of five 6TM variants in mouse, MOR‐1G was quite abundant and conserved from rodent to humans. The 6TM variants are essential for analgesic actions of a novel type of analgesic drugs such as 3‐iodobenzoyl‐6β‐naltrexamide (IBNtxA) that is potent analgesic against a spectrum of pain models without many traditional opiate side effects. The 6TM variants also involve analgesia of delta and kappa opioids and α2‐adrenergic drugs. In the present study, we demonstrate a new function of MOR‐1G in enhancing expression of the full‐length 7TM mu opioid receptor, MOR‐1, using a Tet‐Off inducible system in Chinese Hamster Ovary cells. When co‐expressed, MOR‐1G had no effect on expression of MOR‐1 mRNA, but greatly increased MOR‐1 expression at protein level in a dose‐dependent manner determined by opioid receptor binding and [35S]GTPγS binding. Interestingly, co‐expression of MOR‐1 with MOR‐1G significantly altered mu binding profile for several mu agonists, including M6G, β‐endorphin and dynorphin A, determined by competition assay. We further show that MOR‐1G can physically interact with MOR‐1 through heterodimerization using co‐immunoprecipitation approach, providing a molecular mechanism of how MOR‐1G regulates expression and function of the full‐length 7TM mu opioid receptor.Support or Funding InformationThis work was supported by the National Institute on Drug Abuse (DA042888, DA046714 and DA007242), the Mayday Foundation, the Peter F. McManus Charitable Trust and a core grant from the National Cancer Institute to Memorial Sloan‐Kettering Cancer Center (CA08748).
Isolation and Purification of Functional Amyloid Proteins in Haloferax volcanii
Authors: Sawyer Erica , Kozodoy Lisa , Raimer Heather , Olendzenski Lorraine , Marano Nadia 
Published: April 2020

Haloferax volcanii, an extremophile species of Archaea, is a microorganism that incorporates functional amyloids into its biofilms; these proteins are proposed to contribute to the structural stability of this matrix. We developed a protocol based on the procedure described by Jordal et al. (2009) to isolate these proteins via differential centrifugation. The presence of amyloid proteins was monitored and quantified throughout this process using Thioflavin T, a molecule that can selectively quantify amyloids by fluorescing when bound to their cross‐beta motif. These results showed that a four‐week growth period was ideal and resulted in the highest amyloid fiber yield. The final supernatant contained the majority of the amyloid protein and had percent recoveries ranging from 41.2% to 63.4%, while the final pellet had recoveries from 6.3%‐18.1%. To further improve the efficiency of this procedure, we are utilizing the detergent Triton X‐100 during the isolation process. Preliminary findings show that this detergent slightly increases the amyloid percent recovery. Isolated proteins will ultimately be purified using preparatory SDS‐PAGE for atomic force microscopy imaging, deaggregated into their monomeric forms using formic acid, and sequenced to gain further information about their structure.Support or Funding InformationSLU Fellowship Program & Strading Research Fund
Determining the DNA Binding Preference of the Transcription Factor Optix in Heliconius Butterflies
Authors: Barreto Anthony Rafael Rivera , Martínez José Arcadio Rodríguez 
Published: April 2020

Transcription factors (TFs) are sequence‐specific DNA binding proteins that determine cell state and have been shown to control cell differentiation. Evaluating comprehensive DNA specificity profiles of DNA binding proteins is a nontrivial challenge that hinders our ability to decipher gene regulatory networks. Optix is a homeodomain transcription factor that has been identified as a master regulator of red color patterning in the wings of Heliconius butterflies. However, the direct gene targets of optix and its role in final wing coloration remains to be determined. Because of this, we want to determine the intrinsic DNA‐binding preferences of optix. We have over‐expressed and purified full‐length Heliconius optix. To achieve our goal, we have used Cognate Site Identification by Systematic Evolution of Ligands by Exponential Enrichment with Parallel sequencing (SELEX‐seq). We are currently predicting optix possible binding sites, by using computational analysis. Our results will enable us to bioinformatically predict the genomic targets of optix and decipher its role in wing color patterning and development.Support or Funding InformationNSF EPSCoR Research Infrastructure grant award 1736026Research Initiative for Scientific Enhancement (RISE) 5R25GM061151‐18
Restoration of the Blood‐Spinal Cord Barrier after Spinal Cord Injury through 5‐HT1F Receptor Mediated Mitochondrial Biogenesis
Authors: Simmons Epiphani Ciara , Carroll Seth S. , Schnellmann Rick G. 
Published: April 2020

Vascular and mitochondrial dysfunction are well‐established consequences of spinal cord injury (SCI). Evidence suggests mitigating these dysfunctions may be an effective approach in treating SCI. We propose to restore vascular and mitochondrial dysfunction following SCI through pharmacological induction of mitochondrial biogenesis (MB). We previously reported that 5‐hydroxytryptamine receptor 1F (5‐HT1F) agonism induces MB in the spinal cord and promotes locomotor recovery with drug initiation beginning at a clinically relevant time point of 8h post‐SCI. The goal of this study was to elucidate if MB induction with the newer selective 5‐HT1F receptor agonist lasmiditan can enhance restoration of the blood‐spinal cord barrier (BSCB) after SCI. C57BL/6J mice were subjected to moderate SCI using a force‐controlled impactor‐induced contusion model followed by daily administration of lasmiditan (0.1 mg/kg, i.p.) beginning 1h after injury. Integrity of the BSCB was assessed using Evans Blue dye extravasation. SCI increased dye extravasation at 1, 3, and 7d, and lasmiditan‐treated mice displayed reduced dye accumulation at 7d post‐SCI, suggesting accelerated BSCB recovery. Levels of tight junction proteins occludin and zonula occludens‐1 were decreased in vehicle‐treated mice and returned to sham levels with lasmiditan treatment by d7. These findings led us to investigate if lasmiditan induces MB in endothelial cells. Primary cultures of cerebral microvascular endothelial cells from C57BL6 mice were treated with lasmiditan. A Seahorse Biosciences flux analyzer was used to quantify FCCP‐uncoupled oxygen consumption rate (OCR), a marker of MB. Lasmiditan increased FCCP‐uncoupled OCR, nuclear‐ and mitochondrial‐encoded proteins (PGC‐1a, COX1, TFAM), and mitochondrial number, confirming MB. Lastly, treatment with lasmiditan enhanced endothelial branching morphogenesis and migration, indicating a role of the 5‐HT1F receptor activation in angiogenic pathways. Collectively, these data reveal that 5‐HT1F receptor agonist‐induced MB occurs in endothelial cells and may accelerate restoration of the BSCB after SCI.Support or Funding InformationThis study was supported by the National Institutes of Health National Institute of General Medical Sciences: GM084147 (R.G.S), and the Biomedical Laboratory Research and Development Program of the Department of Veterans Affairs: BX: 000851 (R.G.S.).
Structural Mechanism of Substrate Specificity in Human Cytidine Deaminase Family APOBEC3s
Authors: Hou Shurong , Nalivaika Ellen A. , Myint Wazo , Matsuo Hiroshi , Yilmaz Nese Kurt , Schiffer Celia A. 
Published: April 2020

APOBEC3s proteins (A3s), a family of cytidine deaminases, protect the host cell from endogenous retro‐elements and exogenous viral infections by introducing hypermutations. However, ability to mutate genomic DNA makes A3s a potential cancer source, as has been reported for A3A, A3B and A3H. Despite overall similarity, A3 proteins have distinct deamination activity and substrate specificity. Over the past years, several crystal and NMR structures of apo A3s and DNA/RNA‐bound A3s have been determined. These structures have suggested the importance of the loops around the active site for nucleotide specificity and binding. However, the structural mechanism underlying A3 activity and substrate specificity requires further examination. Using advanced molecular modeling followed by experimental mutational analysis and molecular dynamics (MD) simulations, we revealed the structural basis for DNA binding and relatively lower catalytic activity of A3B compared to the highly similar A3A. We identified Arg211 in loop 1 as the gatekeeper coordinating DNA, and critical residues for nucleotide specificity. We also identified a unique auto‐inhibited conformation in A3B that restricts access to the active site and may underlie lower catalytic activity. Recently, we investigated the structural mechanism for exclusion of RNA from A3G catalytic activity using similar methods. Overall, the comprehensive structural analysis of A3 domains in this project will reveal determinants of substrate specificity and shed light into the biological function of these enzymes, potentially leading to discover inhibitors to benefit cancer therapeutics. In addition, rational engineering of A3 domains to achieve varying catalytic efficiencies or distinct substrate specificity will be greatly beneficial for CPRISR–A3 base editing systems in gene therapy.Support or Funding InformationThis work is supported by the U.S. National Institutes of Health Institute of General Medical Sciences [R01GM118474].
Peripheral cannabinoid CB1 receptors control nutrient‐induced incretin secretion in vivo
Authors: Perez Pedro Antonio , Argueta Donovan , DiPatrizio Nicholas Vincent 
Published: April 2020

Enteroendocrine cells along the intestinal epithelium secrete incretins and related hormones (i.e. GIP, GLP‐1, and CCK‐8) following ingestion of nutrients. Incretins bind and activate their respective receptors on cells located in – but not limited to – the endocrine pancreas, which enhances insulin secretion and maintains a tight window of circulating blood glucose. The ability to adequately regulate circulating glucose levels is lost in patients living with prediabetes and type 2 diabetes. The endocannabinoid system controls food intake and glucose homeostasis, and its activity becomes upregulated in the proximal small‐intestinal epithelium of Western‐diet induced obese mice. Cannabinoid subtype‐1 receptors (CB1Rs) are present in various incretin‐producing cells in the intestinal epithelium (e.g. K, L, I‐cells), yet their function in these cell types are poorly characterized. In the current investigation, we examined the ability for CB1Rs to control incretin secretion in vivo in male C57CL/6N mice. We administered the cannabinoid receptor agonist, WIN 55,212‐2 (WIN, 3 mg per kg), alone and in combination with the peripherally‐restricted neutral CB1R antagonist, AM6545 (10 mg per kg). Incretin secretion was then stimulated via corn oil gavage (0.5 mL 30 minutes after drug treatment) and circulating incretins were quantified by sensitive enzyme‐linked immunosorbent assays from plasma obtained 30 minutes later. Activating CB1Rs with WIN blunted corn‐oil induced secretion of GIP, active GLP‐1 and CCK‐8. Co‐administration of WIN with AM6545 completely reversed the inhibitory actions of WIN on incretin release. Collectively, our results suggest that CB1Rs in the small‐intestinal epithelium control nutrient induced incretin release. The functional consequences of these findings on glucose homeostasis are currently under investigation.Support or Funding InformationAcknowledgementsFunded by the National Institutes of Health, USA (Grant no. DK119498, DK114978, and DA034009)
Modulations in Structure, Dynamics, and Allostery for Emerging Beta‐Lactamase Variants
Authors: Page Richard C. 
Published: April 2020

Antibiotic resistance is a continual and growing threat to human health. The use, and overuse, of antibiotics increases the prevalence of antibiotic resistance observed in the clinic. The concomitant emergence of new beta‐lactamase variants, capable of hydrolyzing a broader library of beta‐lactam antibiotics, is particularly worrisome. The development of new antibiotics and of beta‐lactamase inhibitors for restoring efficacy of current generation antibiotics depends upon a strong understanding of the structures, dynamics, and substrate profiles for clinically‐relevant beta‐lactamases. Here we discuss ongoing biophysical, structural, and biochemical efforts using NMR, X‐ray crystallography, differential scanning fluorimetry, and a range of activity assays. These efforts are increasing our understanding of emerging beta‐lactamase variants and producing custom‐designed beta‐lactamase constructs to enable studies on fundamental mechanisms, stability, dynamics, and substrate interaction profiles.Support or Funding InformationNIGMS R35 GM128595
Molecular and cellular insights into mechanosensation and volume regulation in the nervous system
Authors: Brohawn Stephen 
Published: April 2020

The transduction of physical forces into cellular electrical signals is fundamental to a wide breadth of biology including the classic senses of touch, pain, hearing, and balance; less conspicuous sensations of proprioception, blood pressure, and osmolarity; and basic aspects of neural growth, signaling, and death. In this talk, I will discuss recent work aimed at understanding the basis of mechanosensation and volume regulation in the nervous system. First, I will present a model for the mechanism of membrane tension sensing and physiological roles of a mechanosensitive potassium channel based on a combination of structural, electrophysiological, and localization data. Second, I will present a model for ionic strength sensing and channel gating in a family of volume regulated anion channels based on structural and functional studies.Support or Funding InformationNew York Stem Cell Foundation ‐ Robertson Neuroscience Investigator Award, McKnight Scholar Award, Klingenstein‐Simons Fellowship Award, NIGMS New Innovator Award, Rose Hill Innovator Award
Exploring Hsp70 chaperone interactions with a full‐length substrate
Authors: Saini Sashrika , Clérico Eugenia M. , Meng Wenli , Gierasch Lila M. 
Published: April 2020

Molecular chaperones play a key role in maintaining a healthy cellular proteome by performing protein quality control. Heat shock protein 70s (Hsp70s) are a diverse class of evolutionarily conserved chaperones that interact with short hydrophobic sequences presented in unfolded proteins, promoting productive folding and preventing proteins from aggregation (Clérico et al., 2015). Despite extensive research that reveals a selective promiscuity between the Hsp70 substrate‐binding domain (SBD) and client proteins, there is a paucity of data on the interaction of Hsp70s with large protein clients. To investigate the interaction between Hsp70s and a full‐length substrate, we chose to characterize the binding of the E. coli homolog,DnaK, to the precursor of alkaline phosphatase (proPhoA). proPhoA is an endogenous substrate of DnaK that is unfolded and soluble under reducing conditions, and contains at least five sequences that bind DnaK when presented as short peptides (Rüdiger et al., 1997; Wild et al., 1992). To simplify the experimental conditions we used truncated versions of proPhoA (~100 residues long) that contain 1 or 2 binding sites. We used biophysical techniques to reveal the affinity of binding, and NMR spectroscopy to elucidate the mode of chaperone binding to the proPhoA fragments. We found that there is a hierarchy of affinities among the 5 potential binding sites, and novel cryptic binding sites have been observed. In addition, data suggest there may be a role for residues beyond the canonical five‐residue core sequences. The mechanism of DnaK binding site selection on a substrate containing multiple potential sites and the structural basis of interaction between the chaperone and its respective binding sites on a full‐length substrate will be described.Support or Funding InformationNIH R35 GM118161 MIRA Grant "Protein folding in the Cell: Challenges and Coping Mechanisms" 6/1/16 – 5/31/2021