bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Analysis of DUF1 in Saccharomyces cerevesiae
Authors: Keuls Rachel , Lozzi Brittney , Anderson Philip , Erickson F. Les 
Published: November 2023

The yeast DUF1 gene encodes a highly‐conserved, WD40‐repeat protein that interacts with and activates deubiquitinases (DUBs). DUBs carry out the process of deubiquitination, the removal of ubiquitin from tagged proteins. Animal homologues of DUF1 have been shown to form complexes with DUBs that have a variety of important functions. Human WDR48 activates DUBs that deubiquitinate key subunits in the Fanconi anemia DNA repair pathway. Caenorhabditis elegans WDR48 interacts with Usp46 and stabilizes Glutamate receptors (GLR‐1) to promote changes in locomotion behavior. Previous research has shown that in Saccharomyces cerevisiae, Duf1 interacts with Ubp9 and Ubp13, DUBs involved in the biosynthesis of mitochondrial subunits necessary for respiration. When a DUF1 mutant strain was grown at a higher temperature on a non‐fermentable carbon source, increased petite colony formation was observed. Thus, we hypothesize that Duf1 enhances the yeast's ability to cope with stress. Our interest lies in determining which cellular stress pathways require Duf1 in order to gain insight about its function. By exposing a DUF1 knockout mutant strain to different stress conditions and comparing its growth to the wild type parent strain, phenotypes may be revealed to provide a better understanding of the cellular activities of Duf1. Cell growth experiments were performed in 96‐well plates using a Spectramax i3 plate reader and cell number was plotted against time. Growth rate and maximum cell number were computed in R and differences between the knockout mutant and the wild type parent strain were determined using a pairwise Mann‐Whitney U test. When grown in minimal media, the knockout mutant strain grew faster than the wild type parent strain. The knockout mutant was also able to reach a larger maximum cell number than that of the wild type parent strain in minimal media containing high levels of glucose, a stressful growth situation. Conversely, the wild type parent strain grew better when minimal media was supplemented with high amounts of ethanol. This implies that the action of Duf1 as either a positive or negative regulator is dependent upon the type of stress experienced and that DUF1 may play a role in multiple cellular stress response pathways.Support or Funding InformationThis project is supported by an NSF Major Research Instrumentation grant and Salisbury University's Henson Undergraduate Research Fund.
Optimization of Falling Drop Copper Sulfate Method to Quantify Hemoglobin Concentration
Authors: Sinha Rahul Kumar , Randolph Tim R 
Published: November 2023

Many clinics in underdeveloped countries lack stable electricity, climate control, refrigeration, clean water, and properly trained staff, necessary to implement modern laboratory testing methods. Anemia is a common condition around the world and blood hemoglobin testing has proven to be a significant challenge in these clinics. Even simple methods of anemia assessment like microhematocrit, HemoCue and reflectance instruments require electricity or batteries and are cost prohibitive. This research aims to optimize the Falling Drop hemoglobin method previously developed in our laboratory at Saint Louis University, and in particular to assess the linearity between the descent time of a drop of blood and the hematocrit level of the sample. In accordance with our method, blood was collected in tubes coated with sodium fluoride/potassium oxalate and 44uL was delivered through a modified pipette tip onto a 4.58% solution of copper sulfate in a column created from a 10mL pipette. The descent time of a drop of blood through a column of copper sulfate was measured and recorded. Normal blood was mixed with autologous plasma to create eight sets of hematocrit levels ranging from 15% to 50% in 5% increments. Six sets of eight tubes were created and each hematocrit level was dropped 6 times through a column. A strong inverse correlation was assessed, with the lowest R2 = 0.8461 and the highest R2 = 0.9703. After plotting the mean of each hematocrit level for all 6 of the runs, the R2 = 0.9622. Other best fit models were also evaluated. Therefore, a correlation between blood drop descent time and hemoglobin concentration does exist, and this can be applied to optimize a method to determine the hemoglobin level of a blood sample.Support or Funding InformationRKS is supported by the DeNardo Education and Research Foundation.
UV MUTAGENESIS ENHANCE FUNCTION OF CELLOBIOHYDROLASE I GENE OF ASPERGILLUS NIGER
Authors: Babalola Musa Oladayo , Magbagbeola Olubunmi Abiola , Ebuehi Albert Osaretin , Okochi Veronica Ihuaku 
Published: November 2023

BackgroundThe need for utilizing renewable resources to meet the future demand for fuel and other value added products has increased the attention on lignocellulosic wastes. Lignocelluloses are degraded by cellulase and other lignocellulolytic enzymes produced by fungi and bacteria. Mutagenic agents have been used to achieve improvement of these strains. There is however, paucity of data on the effect of these mutagenic agents on the structure and function of cellulase genes. The aim of this study was to carry out a structural analysis on selected cellulase genes obtained from ultraviolet light mutated Aspergillus niger.MethodsFungi were isolated from decomposing wood wastes. They were genotyped by amplifying the internally transcribed spacer (ITS) regions on their DNA, sequencing the amplicons and analyzing the sequences. They were genetically modified for enhanced cellulase production using ultraviolet rays. RNA of the mutated A. niger was reverse transcribed to complementary DNA (cDNA). The selected genes; aepI, aceI, cbhI and a laccase gene, Lacc1 were amplified and sequenced. The sequences were aligned and compared with referenced sequences which included those of Trichoderma reesei and Phanerochaete chrysosporium. The sequences were also translated into amino acid and in‐silico X‐ray crystallography structure of the active site of the enzyme was constructed using MEGA5 and JPred version of Jalview softwares.ResultsBioinformatics analysis of the sequence of the cbh I gene of the A. niger mutant showed that it's enzyme had strong similarity with compared industrially beneficial fungi; Trichoderma reesei and Phanerochaete chrysosporium. Amino acid changes occurred between the 8th to the 92nd position on the chain‐A of the enzyme. Amino acids residues in its active site had low hydropathy index. Predicted structure of cellulase from A. niger mutant revealed that alteration occurred in the β‐pleated sheets of the enzyme.ConclusionGenetic modification of Aspergillus niger resulted in enhanced cellulase enzyme activity.Support or Funding InformationCentral Research Laboratory, College of Medicine, University of Lagos
The Anti‐Proliferative Effects of Curcumin Derivatives, Dimethoxycurcumin, Bisdemethoxycurcumin and Tetrahydrocurcumin, on DLD‐1 Colon Cancer Cells
Authors: Khatwani Natasha , Adeyeni Temitope , Ezekiel Uthayashanker 
Published: November 2023

Phytochemicals are natural plant‐derived products that show preventative and therapeutic properties against a wide range of human diseases. Studies comparing the effects of Western and Eastern diets on disease outcome have shown that consumption of a diet rich in these phytochemicals proves effective in inhibiting colon cancer. Curcumin, the predominant hydrophobic polyphenolic compound that is isolated from the rhizomes of Curcuma longa, has attracted a lot of attention due to its diverse applications in traditional medicines as a potent anti‐inflammatory, antioxidant, antiangiogenic and anticancer agent. Despite its promising chemotherapeutic activity, preclinical and clinical studies of curcumin highlight its narrow in vivo therapeutic application due to its instability and limited bioavailability in human physiological conditions. For these reasons, we compared the chemotherapeutic effects of three curcumin derivatives, dimethoxycurcumin, bisdemethoxycurcumin and tetrahydrocurcumin, that studies have shown to have increased bioavailability and antiproliferative effects on various cancer cell lines, versus curcumin by measuring their antiproliferative effects on the DLD‐1 colon cancer cell line. The results showed that while bisdemethoxycurcumin (IC50 = 10 μM) showed similar effects and tetrahydrocurcumin (IC50 = 50 μM) showed less potent antiproliferative effects than curcumin, dimethoxycurcumin showed 6‐fold increased potency (IC50 = 2 μM) compared to curcumin. In previous experimental studies on curcumin we observed that the antiproliferative effects of the phytochemical were enhanced in combination with silymarin, the bioactive component of milk thistle. Given this promising synergistic interaction and given the range of results displayed in our current study of the curcumin derivatives, our future experiment will be focused on the comparative studies of the potential synergistic interactions of dimethoxycurcumin, bisdemethoxycurcumin and tetrahydrocurcumin with silymarin.Support or Funding InformationNK and TA are supported by the DeNardo Education and Research Foundation Fellowship.
Determination of HNO‐Derived Modifications on the Cardiac Protein Phospholamban
Authors: Keceli Gizem , Majumdar Ananya , Thorpe Chevon N. , Mahaney James E. , Paolocci Nazareno , Toscano John P. 
Published: November 2023

Congestive heart failure frequently involves reduced cardiac contractility and slowed muscle relaxation. HNO (nitroxyl, azanone), the protonated one electron reduced form of nitric oxide, has received significant attention as a potential heart failure therapeutic. This reactive nitrogen species augments contractility and accelerates relaxation in both normal and failing hearts. In humans, greater than 70% of cytosolic Ca2+ is pumped into the sarco(endo)plasmic reticulum (SR) by SR Ca2+‐ATPase (SERCA2a). The activity of the Ca2+ pump is regulated by a small, transmembrane protein, phospholamban (PLN). Our previous work has shown that HNO modifies PLN and affects SERCA2a activity independent of the β‐adrenergic pathway. We have applied our 15N‐edited NMR method for sulfinamide detection and biochemical approaches to investigate HNO‐derived modifications on PLN. These studies demonstrate that PLN cysteines 46 and 41 are the sites of HNO reactivity. Furthermore, our results indicate that sulfinamide formation is the major HNO‐derived modification in the presence of excess HNO donor, whereas disulfide formation is expected to dominate under physiologically relevant conditions.Support or Funding InformationNational Science Foundationand Cardioxyl Pharmaceuticals
Bacterial Cell Wall Remodeling to Study the Production of Immunoactivating Fragments
Authors: Liang Hai , DeMeester Kristen E , Hou Ching‐Wen , Grimes Catherine Leimkuhler 
Published: November 2023

The human innate immune system is the front line of defense to protect the body from bacterial infection. One mechanism of activation involves various immunoactivating fragments derived from bacterial cell wall engaging the appropriate receptor to trigger an inflammatory response. Muramyl dipeptide (MDP), a small fragment of bacterial cell wall has been shown to have immunoactivating properties via the innate immune receptor Nod2. Deficiency in sensing MDP may cause serious inflammatory bowel disease (IBD) such as Crohn's Disease (CD). However, how MDP and other immunostimulatory fragments are generated is not well understood. The discovery of new immunoactivating fragments is critical to understand the interactions between human innate immune system and the bacteria it encounters (commensal or pathogenic). Here a combination of organic synthesis, biochemistry and genetic engineering approaches were implemented to bioorthogonally label bacterial peptidoglycan. We have synthesized a variety of azide functionalized cell wall intermediates, including: D‐form amino acids and UDP‐sugar substrates. The peptidoglycan biosynthetic enzymes were purified and assayed for substrate promiscuity. As these enzymes showed relaxed substrate specificity, bacterial cells were grown in the presence of the azide containing precursors. The cells were then labeled with a bioorthogonal azide‐alkyne click reaction to link a fluorescent molecule onto the cell wall. The bacterial cell walls were successfully visualized. Using this method, we will further study the interaction of human innate immune cells and fluorophore labeled bacterial cells to reveal new immunoactivating molecules.Support or Funding InformationFunding of this project is provided by Centers of Biomedical Research Excellence (COBRE)
Carbohydrate‐Protein Interactions with Poly‐amido‐saccharides (PASs) as Unique Carbohydrate‐Inspired Polymer Structures
Authors: Chin Stacy L. , Grinstaff Mark W. 
Published: November 2023

Natural polysaccharides are of significant interest in biochemistry research because of their structural diversity and essential roles in metabolism, energy storage, cellular signaling, protein recognition, and other physiological processes. However the synthesis of polysaccharides is challenging due to the presence of many similar functional groups and the large number of stereocenters throughout the polymer structure. In response to the need for reliable approaches to prepare polysaccharides, we reported a synthetic method to prepare well‐defined, enantiopure carbohydrate polymers, termed poly‐amido‐saccharides (PASs), using a controlled anionic polymerization of b‐lactam sugar monomers. Unlike natural polysaccharides, in which sugar units are joined by ether linkages, pyranose units in PASs are linked through the 1‐and 2‐positions by an amide group, which affords PASs with unique structural features and interesting chemical properties. We recently found that PASs 1) possess a helical conformation in their secondary structure, 2) are able to maintain its rigid structure under varying pH, temperature, ionic strength, and denaturing conditions, and 3) are non‐cytotoxic and can be endocytosed across different mammalian cell lines.Our initial investigation indicate that glucose‐derived PAS (glc‐PAS) structures can be recognized by lectin concanavalin A at the same site as glucose through binding activity assays, which suggests the potential for PASs to serve as natural polysaccharide mimics. Based on this preliminary result, we hypothesize that sulfated galactose‐derived PAS (gal‐PAS) structures can potentially interact proteins in the coagulation cascade and posses anticoagulant‐like properties, similarly to heparin. The synthesis and characterization of glc‐PAS, gal‐PAS, and sulfated glc‐PAS polymer structures with via IR, NMR, and GPC will also be discussed.
Differential Regulation of mTORC1 by Amino Acids
Authors: Jewell Jenna L , Guan Kun‐Liang 
Published: November 2023

Organisms sense nutrient fluctuations and respond accordingly to control cell growth. When this process goes awry, the result can be detrimental, often leading to cancer. Central to this process is the mechanistic target of rapamycin (mTOR), a conserved kinase that is the main component of a protein complex termed mTOR complex 1 (mTORC1). mTORC1 activation is frequently observed in human cancers, and therapeutics that target mTORC1 have proven efficacious in clinical trials. Amino acids are the most potent stimuli for mTORC1 activation; however, the detailed molecular mechanisms are obscure, and importantly, the specific amino acids that regulate mTORC1 are unclear. Recently, we discovered that the amino acids, leucine and glutamine activate mTORC1 through two distinct signaling pathways involving the small G‐proteins RagA/B and the adenosine diphosphate ribosylation factor‐1 (Arf1), respectively. Importantly, we identified Arf1 as an essential component that specifically couples glutamine signaling to mTORC1 activation at the lysosome and independent of RagA/B. This is important because cancer cells have a high dependence on glutamine for growth and proliferation. Our results uncover a signaling cascade to mTORC1 activation independent of the Rag GTPases and suggest that mTORC1 is differentially regulated by specific amino acids.Support or Funding InformationCancer Prevention & Research Institute of Texas (CPRIT)
Avocado Oil Improves Mitochondrial Function and Decreases Oxidative Stress in Kidney Mitochondria of Type‐2 Diabetic Rats
Authors: Ortiz‐Avila Omar , del Consuelo Figueroa‐Garcia Maria , Mejia‐Zepeda Ricardo , Cabrera‐Nuñez Faride , Flores‐Ledesma Claudia G , Hernandez‐Esparza Manjury J , Saavedra‐Molina Alfredo , Cortes‐Rojo Christian 
Published: November 2023

Nephropathy is one of the most worrying complications of diabetes because of its direct association with the high mortality rate in diabetic patients. Diabetes is characterized by an increase in ROS generation and impaired mitochondrial dysfunction. Overproduction of mitochondrial ROS has been recognized as the single major event leading to the activation of pathways conducing to diabetic nephropathy. Previously, we have shown that avocado oil prevents kidney mitochondrial dysfunction and decreases ROS production in a rat model of type I diabetes. Avocado oil is a source of MUFA with some potential beneficial effects in health such as improvement of diabetic dyslipidemia, also contains several antioxidants which may modulate ROS production. Despite these antecedents, it is unknown whether avocado oil has the same effects over kidney mitochondrial dysfunction and excessive ROS production in a rat model of type 2 diabetes. To address this issue, avocado oil was administered for 3 and 6 months to Goto‐Kakizaki rats, a lean model of type 2 diabetes. Diabetic rats display a severe impairment in state 3 respiration, leading to 68% and 45.6% decrease in respiratory control ratio (RCR). Avocado oil slightly prevented this effect at 3 months but fully prevent inhibition of RCR at 6 months. Diabetes elicited a 70% decrease in mitochondrial membrane potential at 3 months, with avocado oil slightly preventing this effect. In contrast, at 6 months, there was no difference in this parameter in any experimental group in comparison to the control. Avocado oil led to a decrease of 38% and 70% in ROS production in mitochondria from diabetic rats at 3 and 6 months, respectively, in comparison to untreated diabetic rats. Mitochondria from diabetic rats exhibited a 83% decrease in mitochondrial GSH/GSSG ratio at both 3 and 6 months, indicating a condition of strong oxidative stress; supplementation with avocado oil fully prevented this effect at 3 months while at 6 months, a partial protective effect was seen as mitochondria of diabetic rats supplemented with avocado oil shown a ~40% decrease in this parameter. These results suggest that avocado oil prevented also type 2 diabetes‐induced mitochondrial dysfunction and exacerbated oxidative stress in kidney mitochondria.Support or Funding InformationSupported by a grant from Programa de Investigación 2016 de la Coordinación de la Investigación Científica de la UMSNH (CIC‐UMSNH) (CCR).
Engineering Clonal Human Cell Lines Stably Expressing a Single p73 Isoform
Authors: Cordova Ricardo A. , Gushterova Irena , Carastro L. Michael 
Published: November 2023

The p73 gene is a member of the p53 tumor suppressor family. This gene has two promoters, P1 and P2, which are the transcription initiation sites for the mRNAs encoding TAp73 and DNp73 isoforms, respectively. Recently, we reported that a human colon cancer cell line, Caco‐2, has no detectable expression of either of the two p73 protein isoforms through immunoblotting. In order to facilitate future molecular studies of specific p73 isoform functions as well as to create control samples expressing only one detectable p73 isoform for immunoblotting and other molecular techniques, pCI‐neo mammalian expression vectors encoding one of the four p73 isoform cDNAs (TAp73a, TAp73b, DNp73a, DNp73b) were stably transfected into Caco‐2 cells by electroporation. Clonal populations of transfected cells were selected using antibiotic resistance, isolated and expanded. Clonal cell line populations were used to isolate total RNA using RNeasy Plus Mini Kits (QIAGEN). Total RNA samples were used to synthesize cDNA and perform TaqMan Real Time‐PCR Comparative CT analyses in order to determine the p73 isoforms mRNA expression levels relative to untransfected Caco‐2 cells. Our initial TaqMan Real Time‐PCR data indicate a 6‐fold expression of TAp73 mRNA in clone TAp73a #12 (RQ = 6.1536; RQmin = 2.6054, RQmax = 14.0758), a 25‐fold expression of TAp73 mRNA in clone TAp73b #2 (RQ: 24.9555; RQmin = 23.3701, RQmax = 26.6485), 1‐fold expression of DNp73 mRNA in clone DNp73a #9 (RQ: 1.3434; RQmin = 0.8763, RQmax = 2.0594) and 1800‐fold expression of DNp73 mRNA in clone DNb #2 (RQ: 1850,175; RQmin = 1587.564, RQmax = 2156.226) as compared to untransfected Caco‐2. Based on our TaqMan Real Time‐PCR data, we concluded that these four isolated clonal Caco‐2 cell populations overexpress the transfected p73 mRNA isoform.Support or Funding InformationWe thank The University of Tampa David Delo Research Professor Grant (to L.M.C.) for generous financial support of this project.
Conformational Flexibility of SIKE
Authors: Standen Heather , Bell Ellis , Bell Jessica 
Published: November 2023

Suppressor of IKKe (SIKE)inhibits TBK1 mediated phosphorylation of interferon regulatory factor 3 (IRF3) and misregulation of TBK1 is involved in both autoimmune disorders and cancer. Our work (Marion et al, J.Biol.Chem 288, 18612–23, 2013) demonstrated that TBK1 can phosphorylate SIKE in up to 6 positions, altering its ability to inhibit TBK1. Little however is known concerning the three dimensional structure or stability of SIKE or the effects of phosphorylation. SIKE (his tagged) has been expressed and purified using Nickel NTA affinity chromatography and shown to be homogenous by MALDI‐tof mass spectrometry. To determine the effects of phosphorylation on the conformational flexibility of the protein, SIKE and SIKE‐S6E (where the 6 phosphorylatable serines have been mutated to the phosphomimic, glutamate), and to provide support for homology models of the protein created using Phyre2, limited proteolysis experiments, using immobilized trypsin and tandem mass spectrometry identification of the early cleavage sites, and Hydrogen‐Deuterium exchange studies in conjunction with pepsin cleavage after various periods of exchange are being used.Support or Funding InformationThis work was supported by NIH grant R21A1 107447 to JKB