bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Biosynthesis of the Indolic Acid Moiety from the Antibiotic Nosiheptide
Authors: Badding Edward , Booker Squire 
Published: November 2023

Nosiheptide (NOS) is a highly modified thiopeptide natural product produced by Streptomyces actuosus that belongs to a unique class of thiazole‐containing peptide antibiotics. Recent studies have shown that NOS is as an effective antibacterial agent against a number of pathogenic bacteria, such as methicillin resistant Staphylococcus aureus, penicillin‐resistant Streptococcus pneumoniae, and vancomycin‐resistant enterococci. One key structural feature of NOS essential for its antibacterial properties is an indolic acid ring system characteristic of e‐series thiopeptides. The indolic acid moiety is derived from the amino acid tryptophan. In the first step of the proposed pathway, catalyzed by NosL, tryptophan is converted into 3‐methyl indolic acid (MIA). The remaining steps and enzymes involved in the formation and installation of MIA into the NOS core have not yet been definitively determined, but are believed to be catalyzed by the action of four enzymes: NosI, NosJ, NosK, and NosN. The research herein uses isothermal titration calorimetry (ITC), high‐pressure liquid chromatography (HPLC), mass spectrometry, and bioinformatics to delineate the exact roles of each of the enzymes in the biosynthesis of the indolic acid moiety. We show that NosJ is an acyl carrier protein that is post‐translationally modified with a 4′‐phosphopantetheine group, and that NosI catalyzes the ATP‐dependent adenylation of MIA. Upon incubation of NosI with ATP and MIA in the presence of NosJ, we observe transfer of MIA to NosJ. We believe that NosK then catalyzes the transfer of MIA from NosJ to the side‐chain of a cysteinyl residue of NOS. NosN, annotated as a radical S‐adenosylmethionine (SAM) methylase, subsequently attaches a C1 unit—suggested to be a methyl group—to C4 of the indolic acid moiety and then connects the C1 unit to Glu 43 of the macrocyclic core, completing the synthesis of NOS. We are particularly interested in understanding how NosN carries out this reaction. Preliminary ITC experiments show that two SAM molecules bind to the enzyme simultaneously. We suggest that one SAM molecule is converted into a 5′‐deoxyadenosyl 5′‐radical that initiates a novel methylation reaction by abstracting a hydrogen atom from the methyl moiety of the second SAM molecule. These studies shed new light on novel mechanisms for methylating unactivated carbon centers.Support or Funding InformationThis work was supported by NIH (GM103268), the President's Undergraduate Research Fund (Penn State), and the Rodney Erickson Discovery Grant (Penn State).
Investigating Mycobacteriohage‐Host Protein Interactions
Authors: Neevel Andrew , Chang Gloria , VandenBergh Wessel , McDonough Virginia , Stukey Joseph 
Published: November 2023

Mycobacteriophages are viruses that infect bacterial cells of the genus Mycobacterium. With more than 400 mycobacteriophage genomes sequenced and available in GenBank, they represent the largest collection of sequenced phages that infect a single host (Mycobacterium smegmatis). They possess a multitude of unfamiliar or novel genes – genes encoding protein sequences that do not resemble any previously studied proteins – and thus encode products with functions not readily predicted. We hypothesized that some of those genes encode products that interfere with the normal metabolism of the host cell, possibly through specific phage‐host protein‐protein interactions, and thus have a role in enabling phage infection. Further, we predicted that those gene products when expressed alone in host cells would still be toxic and impair cell growth. We have investigated unfamiliar genes in two genetically distinct mycobacteriophages, Pumpkin and Vix, and have identified 4 single genes (Pumpkin_115, Pumpkin_119, Pumpkin_142, Vix_80) and several small genomic regions (Pumpkin gene segment 130–133 and Vix gene segments 65–66, 68–72, 87–88) that are cytotoxic to M. smegmatis. We are taking a multi‐prong approach to further identify the specific functions associated with these genes and their products and to determine their roles in the infection process: 1) we have identified mutants of M. smegmatis that are resistant to the expression of those genes, 2) we are using E. coli‐expressed phage genes to screen for interacting host proteins, 3) we are collecting microscopy data that could identity phage interruption of normal cellular function, and 4) we are in the process of deleting these genes from the phage genome to determine the effect on infection. 24‐hour expression of individual cytotoxic phage genes in M. smegmatis resulted in a significant increase in mean host cell length and some subtle effects on cell shape. Ongoing analysis of the mutants has identified a common mechanism of resistance to distinct phage gene expression, while protein‐protein interaction studies have not yet identified a potential host target involved in translation.Support or Funding InformationSupported by a Jacob E. Nyenhuis Faculty Development Grant and an HHMI Faculty Research Award
Regulation of T cell Activation By Herpes Simplex Virus 1 Protein Us3
Authors: Pan Shuang , Wu Songfang , Liu Yaohui , Zhang Liming , Leng Qianli , Chen Da , Yang Mengmeng , Wang Jiyan , He Bin , Zhang Cuizhu , Cao Youjia 
Published: November 2023

Herpes simplex virus 1 (HSV‐1) is the most prevalent human virus that infects a variety of cell types in latent and lytic states. Previous studies demonstrated that HSV‐1 productively infected human T cells and inhibited T cell receptor (TCR) mediated cell signaling. However, the precise way of virus‐host interactions remains to be solved. Here, we report that HSV‐1 encoded Us3 protein interfered TCR signaling and interleukine‐2 production by targeting the linker for activation of T cells (LAT). Us3 associated with LAT and sequestered the later from TNFR‐associated factor 6 (TRAF6), which plays important role in the activation of LAT. This study unveils a mechanism by which HSV‐1 intrudes early events of T cell activation, and may help to answer how HSV escapes from host immune surveillance.Support or Funding InformationThis work is supported by the National Natural Science Foundation of China 81171556, and 31370862.
Penetration of Staphylococcus aureus Biofilm by the Cinnamon‐Derived Compound, Cinnamaldehyde
Authors: Peach Aaron Preston , Heuertz Rita 
Published: November 2023

Biofilms have become increasingly problematic in efficacious treatment of patient infections due to their tenacious survival, association with chronic and medical device‐associated infections, and resistance to antimicrobial treatments. Of grave concern is the observation that biofilm is frequently a contributory component of antimicrobic resistance and treatment failure in patient infections. Review of the literature reveals that many plant‐derived compounds have anti‐bacterial properties; however, biofilm effects have not been reported. Staphylococcus aureus is known for its multi‐drug resistant strains (MRSA) and plant‐based therapy may be appropriate for treatment of antimicrobic‐resistant bacteria. Our laboratory identified that the cinnamon‐derived compound, cinnamaldehyde, inhibited biofilm production by MRSA in a concentration‐dependent manner. To further this line of study, a penetration assay was developed to determine if cinnamaldehyde was able to penetrate biofilm produced by S. aureus. Briefly, a biofilm‐producing strain of S. aureus (ATCC 29213) was grown on disks of nitrocellulose (pore size 0.45 μm) or Whatman 1 filter paper (pore size 11 μm) and positioned onto Mueller Hinton agar plates streaked with a different S. aureus (ATCC 25923) so as to produce a lawn of growth after incubation. Cinnamaldehyde (at different concentrations) was placed onto the top of the biofilm‐coated disks and plates were incubated. If cinnamaldehyde was able to penetrate the bacterial biofilm on the disk, then it inhibited the lawn of S. aureus growth. Assay development results indicated that application of cinnamaldehyde directly to ATCC 25923 S. aureus inhibited its lawn of growth and that nitrocellulose and filter paper were both acceptable for use in the penetration assay. Early studies suggested that cinnamaldehyde penetrated the biofilm layer: these studies are on‐going. With many infections being associated with biofilms, it is timely to focus on plant‐derived compounds as antimicrobial agents.Support or Funding InformationFinancial support for AP was received from the DeNardo Education and Research Foundation.
Characterization of a Halotolerant Bacterium Isolated from Soil in Suburban Detroit
Authors: Sleda Melissa , Moricz Bridget , Zwiesler‐Vollick Julie 
Published: November 2023

During cold weather, salt is sprayed on roads and sidewalks to prevent ice formation. We sought to determine if this excess salt affected the soil microbial community by increasing the presence of halotolerant or halophilic microbes. We collected a soil sample and isolated a single bacterial colony using nutrient agar plates supplemented with 5% NaCl. The microbe was able to grow indicating an ability to tolerate NaCl. To test if NaCl was required, we grew it on Nutrient Agar without additional NaCl. This microbe was able to grow at least as well without 5% NaCl, indicating that it is halotolerant but does not thrive in the presence of NaCl (halophilic). This bacterial isolate was characterized using multiple staining methods and differential/selective media. We also studied the metabolism of this microbe with respect to its ability to utilize various nutrient sources both in the presence and absence of oxygen. Genomic DNA was extracted and used to amplify the 16s ribosomal RNA gene. This was then sequenced and compared to the 16s RNA bacterial and archaeal database at the National Center for Biotechnology Information (NCBI) to further aid in identification. We were able to isolate a halotolerant microbe showing the presence of a microbe with salt tolerance in the community of salt‐treated soil. The characterization of this microbe will be discussed.Support or Funding InformationFunded by the Department of Natural Science at Lawrence Technological University.
TARGETING TRAF6‐PAK1 SIGNALING LIMITS PROSTATE TUMOR GROWTH AND TGF&[Beta]1‐INDUCED PROSTATE CANCER CELL EPITHELIAL TO MESENCHYMAL TRANSITION
Authors: Somanath Payaningal R , Al‐Azayzih Ahmad , Gao Fei 
Published: November 2023

Recent studies demonstrate the dual role of transforming growth factor beta (TGFβ) in prostate cancer. It is not clear how TGFβ1 that suppresses early prostate tumor growth induces epithelial‐to‐mesenchymal transition (EMT) in advanced stages. We studied if P21‐activated kinase1 (Pak1), a serine‐threonine kinase that mediates cytoskeletal remodeling is necessary for the TGFβ1 induced prostate cancer EMT. Effects of TGFβ1 on control prostate cancer PC3 and DU145 cells and those with Pak1 inhibitor IPA 3 and siRNA mediated Pak1 inhibition were tested for prostate tumor xenograft in vivo and EMT in vitro. TGFβ1 inhibited PC3 tumor xenograft growth via activation of P38‐MAPK and caspase‐3, 9. Long‐term stimulation with TGFβ1 induced PC3 and DU145 cell scattering and increased expression of EMT markers such as Snail and N‐cadherin through tumor necrosis factor receptor‐associated factor‐6 (TRAF6)‐mediated activation of Rac1/Pak1 pathway. Selective inhibition of Pak1 using IPA 3 or knockdown using siRNA both significantly inhibited TGFβ1‐induced prostate cancer cell EMT and expression of mesenchymal markers. Our study demonstrated that TGFβ1 induces apoptosis and EMT in prostate cancer cells via activation of P38‐MAPK and Rac1/Pak1 respectively. Our results reveal the potential therapeutic benefits of targeting TGFβ1‐TRAF6‐Pak1 pathway for advanced‐stage prostate cancer.Support or Funding InformationFunds were provided by the UGA‐Research Foundation, Wilson Pharmacy Foundation, The American Legion, and the NIH (R01HL103952) grants to PRS. Al‐Azayzih was supported by pre‐doctoral fellowship from Jordan University of Science and Technology. This material is the result of work supported with resources and the use of facilities at the Charlie Norwood VAMC, Augusta, GA.
Overexpression of basonuclin 2 promotes carcinogenesis in basal cell carcinoma
Authors: Douhne Timothy , Cornett Jonathan C 
Published: November 2023

Basal cell carcinoma (BCC) is the most common form of human skin cancer. To identify driver genes in BCC tumorigenesis, we carried out a piggyBac transposon mutagenesis screen in mice. Basonuclin 2 (Bnc2) was identified as a gene that, when overexpressed in the skin, likely contributes to the formation of basal cell carcinomas. However, the mechanism by which Bnc2 acts to promote tumor formation is unknown. To gain insight into its role in normal cells, we overexpressed Bnc2 in human keratinocytes. Bnc2 overexpressing cells showed increased viability compared with control cells. In addition, Bnc2 overexpression caused reduced expression of differentiation markers in human keratinocytes. Taken together, our results suggest that Bnc2 acts as a novel oncogene in BCC progression by preventing differentiation and cell death in Bnc2 overexpressing keratinocytes.Support or Funding InformationThis work was supported by a Ledford scholarship from the Appalachian College Association.
Anti‐proliferative Effect of Curcumin is Mediated by Down‐regulation of Polycomb Complex Protein, BMI1
Authors: Adeyeni Temitope Adetola , San KayKay , Ezekiel Uthayashanker 
Published: November 2023

BMI1 encodes a 37‐kDa protein that is a key regulatory component of PRC1 (polycomb regulatory complex‐1). BMI1 functions as an oncogene, induces cell growth and promotes tumor growth in in vitro and in vivo animal models. BMI1 is currently recognized as a gene of interest in terms of pharmaceutical drug development. Curcumin, a major phytochemical in turmeric, inhibits the proliferation of many types of cancer cells, both in vitro and in vivo. Previously, we observed that DLD‐1 colorectal cancer cells had a suppressed BMI1 expression following curcumin treatment. In addition, there was an inhibition of cell proliferation. This statistically significant finding indicates that curcumin potentially inhibits DLD‐1 cell proliferation by targeting BMI1. Curcumin has potential to be used in colorectal cancer cell therapy, however, it has been reported to have poor bioavailability. The purpose of this study is to determine whether or not modified curcumin compounds (dimethoxycurcumin, bisdemethoxycurcumin) and curcumin metabolite (tetrahydrocurcumin), with a higher bioavailability, have the same mechanism of action as curcumin in DLD‐1 colorectal cancer cells. DLD1 cells were treated with curcumin, dimethoxycurcumin, bisdemethoxycurcumin, and tetrahydrocurcumin. After 48 hours, BMI1 gene expression and protein levels will be measured. Preliminary results suggested that dimethoxycurcumin reduced BMI1 protein expression more than curcumin.Support or Funding InformationTAA and KS are supported by the DeNardo Education and Research Foundation.
HER2+ breast cancer metastasis mediated by E2F2 copy number changes
Authors: Andrechek Eran 
Published: November 2023

The E2F family of transcription factors is classically known to regulate G1 to S‐phase transition in cell cycle. Recently, other functions have emerged including roles for E2Fs in cancer, specifically HER2+ breast cancer. We have recently shown that a loss of E2F1 or E2F2 in the MMTV‐Neu mouse model leads to a decrease in tumor metastasis. It is not known what mechanistic roles specific E2Fs are playing in this process. To address this, gene expression of tumors from MMTV‐Neu transgenic mice of wild type and E2F2 knockout backgrounds were analyzed through ACE (Analysis of CNAs by Expression data) to identify regions of chromosomal gain and loss. Conserved mouse and human copy number changes were identified using the TCGA data. These events were screened for effects on patient outcomes through the application of a predictive gene expression signature. An average of 8.8 amplification or deletion events were observed in Neu induced tumors compared to 21.8 events in similar tumors from an E2F2 knockout background (n=15, p<.01). In the E2F2 KO samples, a consistent deletion was found on chromosome 11qD (human chromosome 17q21.33). qPCR analysis of chromosome 11D indicated a significant copy number decrease in MMTV‐Neu E2F2 KO (p<.05). Importantly, further analysis revealed that in 25% of HER2+ve patients there was an amplification of this same region. Strikingly, the presence of this amplification event is associated with a worse metastasis free survival (p<.001) and high E2F2 activity (p<.05). CRISPR mediated knockout studies to determine effects on tumor cell migration in vitro and tumor metastasis in vivo for key genes, including Col1a1 and CHAD, in this amplicon reveal a role in migration. Preliminary results show marked effects on metastatic capacity with knockouts of genes within this amplicon. These results strongly suggest that the E2F family of transcription factors contributes to metastasis partially through copy number changes of regions regulating metastasis in both mouse models and human breast cancer. These findings will have an important impact in understanding the interplay between transcription factors, genomic instability, and tumor metastasis.Support or Funding InformationNIH R01CA160514
A Comparison of SIKE and WIT 3 Structure and Dynamics
Authors: Machek Megan , Minzer Ian , Standen Heather , Kim Seung‐Hwan , Latzer Joachim , 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 of SIKE. SIKE shares significant homology with WIT3 (known to be involved in wound healing) including several of the phosphorylation sites. It is unknown if these sites cause differences in structure and function. SIKE and WIT3 (both his tagged) have been expressed and SIKE purified using Nickel NTA affinity chromatography and shown to be homogenous by MALDI‐tof mass spectrometry. 3Dimensional models of SIKE and WIT3 and various phosphoforms have been constructed using Phyre2 and subjected to molecular dynamics analysis to further explore structural and dynamic similarities and differences between SIKE and WIT3. In an attempt to validate the models limited proteolysis and Hydrogen Deuterium exchange studies are underway.Support or Funding InformationThis work was supported by NIH grant R21A1 107447 to JKB
The Effects of Trifluoroethanol on the Helical Structure of SIKE
Authors: GarciaDuBar Selena , 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 of SIKE. SIKE (his tagged) has been expressed and purified using Nickel NTA affinity chromatography and shown to be homogenous by MALDI‐tof mass spectrometry. To examine the structure of SIKE we have performed two dimensional proton NMR experiments and Circular Dichroism spectroscopy and compared the results with both secondary and tertiary structure predictions. 2D NOESY spectra obtained using several mixing times in general revealed little stable structure in the molecule as detected in the NH‐NH cross peak region, suggesting the protein may contain significant disordered regions. CD spectra suggest significant alpha helix content and the possibility of coiled coil regions of structure. Addition of Trifluoroethanol, known to stabilize alpha helical structures, led to increased CD signal at 222nm. Using a range of TFE concentrations optimal stabilization of helical structure in SIKE allowed comparison with helical secondary structure predications.Support or Funding InformationThis work was supported by NIH grant R21A1 107447 to JKB
PTH Regulation of CEBP‐delta Expression in MAPK Phosphatase‐1 (MKP‐1) Null Osteoblasts from Male and Female Mice
Authors: Datta Nabanita S , Vengalil Nathan , Chukkapalli Sahiti 
Published: November 2023

Age‐related bone loss is associated with a gradual decline in bone homeostasis characterized by increased bone resorption than formation. The mechanisms of osteoblast bone formation are incompletely understood. Parathyroid hormone (PTH) is an important regulator of bone mass and controls osteoblast (the bone forming cells) and osteoclast (the bone resorbing cells) function. MAPKs are negatively regulated by a family of dual‐specificity phosphatases known as the MAPK phosphatases (MKPs). Previous studies from this laboratory have shown that female MKP‐1 knockout (KO) mice display osteopenia, while male KO mice show osteopetrosis. Insulin‐like growth factor‐1 (IGF‐1) plays an important role in promoting osteoblastic differentiation and PTH anabolic action. CCAAT/enhancer‐binding protein delta (CEBP‐delta), a transcription factor, is an upstream regulator of IGF‐1. In order to dissect the molecular mechanism of PTH action on MKP‐1 and sexual dimorphism, this study examined the expressions of CEBP‐delta using primary osteoblasts from wild type (WT) and MKP‐1 KO mice. Calvarial (skull) osteoblasts from 13–16 week old animals were isolated and differentiated for 7‐ days with ascorbic acid. The cells were then treated with vehicle (control) or PTH (100 nM) for 1–2 hour and harvested. Total cellular RNA was isolated and quantitative real‐time PCR was performed. Results indicated sexual dimorphism on CEBP‐delta expression following PTH treatment in both sexes and genotypes. PTH increased 3 to 6‐fold CEBP‐delta expression in KO males but not in WT males. In contrast, 2 to 4‐fold increase in CEBP‐delta expression was noted in WT females compared to 1.5‐fold in KO animals. These findings suggest involvement of CEBP‐delta expression, at least in part, in increased IGF‐1 expression observed in osteoblasts in the absence of MKP‐1 in males. Taken together, our findings provide new insight into the role of MKP‐1 in CEBP‐delta/ IGF‐1 pathway in osteoblast and sexual dimorphism in bone anabolic action.Support or Funding InformationWayne State University Funding for Research and Education (FMRE)