bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identifying Regulatory Targets of the Small RNA MtlS in Vibrio cholerae
Authors: Mendez‐Contreras Sabrina Isabel , Tsao‐Wu Maya , Liu Jane 
Published: June 2021

In the facultative pathogen Vibrio cholerae, MtlS is a small RNA (sRNA) transcribed antisense to the 5′ untranslated region of mtlA, which codes for the transporter of the sugar alcohol mannitol. MtlS works in cis as a translational repressor of mtlA and it is postulated that the sRNA allows the bacteria to respond rapidly to changes in environmental carbon sources. Because bacterial sRNAs can target multiple genes, we hypothesized that MtlS may regulate other genes in addition to mtlA. Previous work in the lab used RNA target‐prediction programs and mass spectrometry‐based proteomics to identify additional MtlS targets. The results identified 6 potential targets: VCA1045 (mtlA), VCA0820, VCA1046, VC1560, VC1899, and VC2683. The objective of this study was to determine if the putative targets were post‐transcriptionally regulated by MtlS. We constructed gfp translational reporter fusions of the candidate genes in a V. cholerae ΔmtlS strain carrying either a control vector or a plasmid ectopically expressing mtlS. An mtlA‐GFP fusion served as a positive control and exhibited the expected down‐regulation of gfp expression when mtlS was expressed. We have tested reporter fusions to all the candidate genes for regulation by MtlS by monitoring total cell fluorescence and western blot analysis. In an experiment done in duplicate in the presence of MtlS, during stationary phase growth we observed 20% down‐regulation of VC1899, encoding a protein with homology to nucleic acid‐binding proteins. In an experiment done in duplicate, we have also observed 30% logarithmic and stationary phase downregulation of VCA1046, encoding the mannitol dehydrogenase mtlD, in the presence of MtlS. Results based on another experiment conducted in duplicate indicate 20% logarithmic and stationary phase down‐regulation of VC1560, encoding a protein with homology to a catalase‐peroxidase, in the presence of MtlS. Tests with VC2683, encoding a putative cystathionine gamma‐synthase, have been inconclusive, as both down‐regulation and up‐regulation by MtlS has been observed; further experiments will need to be conducted with the VC2683‐gfp fusion. The VCA0820‐gfp fusion does not exhibit any fluorescence, in the presence or absence of MtlS. This work positively contributes to the notion that a single sRNA can have multiple regulatory targets. Ultimately, this work will expand our knowledge on MtlS's role in helping V. cholerae adapt quickly to changes in environmental carbon sources and on sRNAs in general.Support or Funding InformationNIH R15 AI090606This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression and purification of the antigen binding fragment of the germline‐encoded precursor to the murine anti‐S1P metalloantibody in the baculovirus‐insect cell system
Authors: Farokhi Elinaz 
Published: June 2021

Sphinosine‐1‐phosphate (S1P) is a biologically active lipid that plays a signaling role in angiogenesis and in tissue remodeling after cardiac ischemia. In recent years, antibodies that target biologically active lipids have been studied as promising therapeutic agents. We determined the 1.9 Å x‐ray crystal structure of a humanized version of the murine anti‐S1P antibody, and reported the novel finding that it employs two bridging calcium ions in binding to its lipid antigen. In order to study the chemical properties of this novel metalloantibody, we are developing methods for recombinant expression and purification. Previously, germline‐encoded light and heavy immunoglobulin genes that served as precursors to the mature anti‐S1P antibody have been engineered into a plasmid vector for recombinant expression in E. coli. Recovery of soluble protein required that both light and heavy genes be directed to the periplasm of the Gram‐negative bacteria through implementation of PelB signal peptides on both genes in a coexpression plasmid. Unfortunately, the yield for the recombinant antibody was low and insufficient for further analyses. Curiously, the affinity chromatography purified protein partitions into two well resolved pools upon further purification by size exclusion chromatography. Sf9 insect cells infected with recombinant baculoviruses have been successfully used to express high yield secreted proteins. Selection of appropriate promoters and signal peptides has been shown to significantly affect protein yield. We employed the pFastBac Dual vector system in which one gene is expressed under the control of the AcNPV polyhedrin promoter while the other is under the control of the AcNPV P10 promoter and the gp64 signal peptide to express the histidine‐tagged heavy (VH and CH1 domains) and light chains of the metalloantibody precursor. The protein concentration is 12 mg/ml in 500 ml suspension insect cells. As part of our efforts to determine whether the incorporation of metals arises during affinity maturation of the antibody or if it is inherent to the germline encoded sequence and as a starting point to study the chemical potential of metalloantibodies, we used ICP‐MS to determine the Ca concentration in samples treated with EDTA. The concentration of Ca in the Fab antibody was significantly higher than the negative control antibody and the buffers. Our next step is to mutate residues that were previously shown to coordinate metals in the anti‐S1P antibody to introduce novel metal binding potential into an antibody scaffold.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Demonstration of the contributions of pulmonary CYPs to naphthalene‐induced airway toxicity using lung‐Cpr‐null mice
Authors: Kovalchuk Nataliia , Zhang Qing‐Yu , Van Winkle Laura , Ding Xinxin 
Published: June 2021

Naphthalene (NA) is an omnipresent air pollutant and respiratory toxicant. NA induces cytotoxicity in airways following bioactivation by CYP (P450) enzymes. Previous studies suggested that liver and lung are both capable of bioactivating NA in vitro and in vivo. However, direct evidence for a specific role of pulmonary P450 enzymes in mediating NA‐induced airway toxicity, particularly upon exposure to NA via the systemic route, is still lacking.The aim of this study was to examine the specific contribution of pulmonary CYPs to airway toxicity of NA using a lung‐Cpr‐null mouse. We predicted that lung epithelial cells that lack Cpr expression, which would have little microsomal P450 activity, would be resistant to NA‐induced cytotoxicity. Cre‐mediated deletion of Cpr in the lung‐Cpr‐null mouse occurred in a substantial proportion of the Club cells, which, before undergoing Cpr deletion, had high ability to bioactivate NA. Under the conditions used, doxycycline‐induced deletion of Cpr occurred in 66% of Club cells in proximal airways and 86% of Club cells in distal airways, based on the results of a dual immunofluorescence study. Levels of NA and NA‐GSH (a biomarker of NA bioactivation) were similar in the plasma of lung‐Cpr‐null mice and their control littermates after administration of a single bolus dose of NA (200 mg/kg). Histological examination of airways revealed that NA‐induced damage of epithelial cells is focal and mild in lung‐Cpr‐null mice compared to severe exfoliation of epithelial cells in most airways in NA‐treated control mice. Moreover, the number of proliferating (BrdU‐positive) airway epithelial cells was 4‐fold lower in NA‐treated lung‐Cpr‐null mice than in NA‐treated control littermates.These results confirm that CYPs in the lung can mediate NA‐induced airway epithelial damage in vivo.Support or Funding Informationsupported in part by NIH grant ES020867This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High‐ throughput Carbonic Anhydrase Activity and Inhibitor Screening Assays
Authors: Hazra Suratna , Tchaga Grigoriy , Yan Gordon July 
Published: June 2021

Carbonic Anhydrase (CA) is a zinc based metalloprotease present in at least 14 different isoforms in mammals. It catalyzes the reversible hydration of carbon dioxide to form bicarbonate and proton, thus playing an important role in pH and CO2 homeostasis. Changes in CA activity are associated with various diseases such as glaucoma, type II diabetes mellitus, lung and liver diseases etc. As CA is also an important target for cancer therapy developing high‐throughput carbonic anhydrase activity and inhibitor screening assays is very important.Carbonic anhydrase activity or carbon dioxide hydration rate is widely measured by using potentiometer or a stopped‐flow instrument as reflected by the change in pH over time. However, this method is not suitable for high‐throughput assay. Consequently, in BioVision, Inc. we have launched the first high throughput Carbonic anhydrase activity assay kit, detecting the esterase activity of this enzyme. This kit utilizes the zinc‐hydroxide mechanism for carbon dioxide hydration and hydrolysis of an ester substrate resulting in a release of a chromophore signal that is proportional to the enzyme activity.Support or Funding InformationBiovision Inc.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification and Characterization of Bioactive Cyclotides in Viola odorata using PepSAVI‐MS
Authors: Parsley Nicole C. , Kirkpatrick Christine L. , Hicks Leslie M. 
Published: June 2021

Antimicrobial resistance (AMR) is present in every country worldwide and has a direct impact on global health, agriculture and economy. Treatment of common bacterial, viral and fungal infections is becoming increasingly challenging as new mechanisms for AMR develop, accelerated by the misuse and overuse of antimicrobial drugs. Botanically‐derived bioactive peptides are an attractive solution to the ongoing deficit of new antimicrobial compounds, with the ability to be highly selective, potent, and inexpensive to produce on a large scale. Our lab has developed the PepSAVI‐MS (Statistically‐guided bioActive Peptides prioritized VIa Mass Spectrometry) pipeline, an adaptable method for the analysis of natural product libraries to rapidly identify bioactive peptides. Using PepSAVI‐MS to screen the cyclotide‐rich botanical species Viola odorata against a variety of human and plant pathogens, we have identified several putative, uncharacterized cyclotides. Cyclotides are a class of cyclic, disulfide‐rich, plant‐derived peptides that boast a diverse range of potent antimicrobial bioactivities, indicating a highly stable, evolvable scaffold. Additionally, we have identified novel bioactivities of a known nematocidal V. odorata cyclotide, Cycloviolacin O8 (cyO8), including anticancer activity against PC3 prostate, MDA‐MB‐231 breast, and OVCAR ovarian cancer cell lines, and for the first time in literature, cyclotide antifungal activity against the common fungal pathogen Fusarium graminearum. Furthermore, to address the identity of the putative, traditionally difficult‐tosequence V. odorata cyclotide species, we demonstrate a proof‐of‐principle 193 nm ultraviolet photodissociation (UVPD) mass spectrometry approach using cyO8 to elucidate the fragmentation efficiency of this information‐rich technique.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of the nuclease resistance of DNA four‐way junctions.
Authors: Moorhead Gaston , Huang Raymond , Kebede Yonatan , Smith Andrew , Estevez Luis , Troisi Maria , Bell Anthony Jerome 
Published: June 2021

Oligodeoxynucleotides (ODN) offer great promise as therapeutics due to their high binding specificity, low toxicity and ease of synthesis. Hence, it is not surprising that ODNs are being developed as antigene and antisense reagents. ODNs can also control gene expression by targeting proteins. One popular approach uses double stranded DNA as transcription factor decoy sequences that are directed against transcription factors such as NF‐kB and specificity protein 1 (Sp1). More recently, researchers have used a similar approach to target proteins in the extracellular matrix. In these studies, nucleic acids are directed against the damage‐associated molecular pattern (DAMP) molecule, High Mobility Group B1 (HMGB1). We hypothesize that ODNs based on DNA four‐way junctions (4WJs) can be used to target HMGB1. To enhance the ex vivo stability of 4WJs, the helical termini of a DNA junction are modified to generate the ODN, i‐J1. The objective of this study is to investigate the nuclease resistance of i‐J1, a control 4WJ (J1) and the intramolecular 4WJ, J4 against three nucleases. The three nucleases are: DNase I, Exonuclease III (Exo III) and bacteriophage T5 Exonuclease (T5 Exo). DNase I cleaves dsDNA and ssDNA in a largely non‐specific manner to generate mono‐ and oligodeoxyribonucleotides. Exo III has expanded functionally; the enzyme acts as a 3′®5′ exonuclease, 3′‐repair diesterase, 3′‐phosphomoesterase and ribonuclease. Here, we focus on the 3′®5′ exonuclease activity of Exo III. T5 Exo hydrolyzes dsDNA and ssDNA in a 5′®3′ direction to release mono‐, di‐, tri‐nucleotides and oligonucleotides. The nuclease protection data shows that i‐J1 and J4 have significantly higher nuclease than J1 against DNase I and Exo III. We suspect the increase in nuclease resistance in linked to changes in the minor groove dimensions of i‐J1 and J4. A binding model is presented to explain the increase in nuclease resistance of i‐J1 and J4.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cleaving Glutathione S‐Transferase from a B‐cell Receptor Protein to Improve Interactions as seen in EMSAS
Authors: Egbuchulam Angela , Lares Monica 
Published: June 2021

The purpose of my project is understanding the specific amino acid interactions for the B‐cell Receptor, BAFF‐R, protein interacting with a RNA aptamer. A RNA aptamer is a peptide that binds to a molecule of interest. We had to express our protein with a GST tag. We used different lab skills, such as gel shift assay, to investigate the interaction between the BAFF‐R and its RNA aptamer. However, the results were inconclusive as we saw an unexpected binding pattern. We hypothesize that cleaving the Glutathione S‐Transferase (GST) tag using thrombin will provide for our expected binding pattern in an EMSA.Support or Funding InformationFunded by Office of Undergraduate Research in Sonoma State University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Searching Protease Inhibitors by a Phage Display Kunitz‐Type Library.
Authors: da Silva Gustavo Felippe , de Lourdes Borba Magalhães Maria , de Moraes Julio Cesar , Echeverri Lina Maria Salazar 
Published: June 2021

Phage Display is a powerful tool for identification of protein ligands against a myriad of targets. In our study, we selected a small protein member from the Kunitz family to serve as a scaffold in the MIII 13 phage coat protein. The protein, originally a scorpion toxin that inhibits a potassium‐dependent voltage channel, has 33 residues composed of two beta sheets and an alpha helix stabilized by disulfide bonds. After cloning the toxin gene into an M13 phagemid, we used Kunkel's mutagenesis, an additional uracil‐DNA glycosylase treatment, and rolling circle amplification (RCA) procedures to produce a synthetic Kunitz‐type phage display library. We drove the mutations in specific residues within two specific loops as well as within the alpha helix of the scaffold protein, keeping the cysteines intact. We were able to produce a library of over a million different proteins. To validate the ability of this library to bind specific targets, trypsin was used as target. Since other studies have proved Kunitz's natural proteins as protease inhibitors, we expect our displayed Phage protein library to generate new potent and specific protease inhibitors for medical as well as biotechnological purposes.Support or Funding InformationThis work was supported by “State University of Santa Catarina” (UDESC) and Fundação de Amparo a Pesquisa e Inovação do Estado de Santa Catarina (FAPESC).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pentachloropseudilin inhibits TGF‐β signaling by promoting TGF‐β type II receptor degradation via non‐raft internalisation
Authors: Chung Chih‐Ling 
Published: June 2021

Transforming growth factor beta 1 (TGF‐β) is a multifunctional cytokine that regulate several cellular processes including cell proliferation, differentiation and apoptosis. In addition to normal physiological function, TGF‐β has been reported that involved in many diseases such as tumor progression and fibrosis. Therefore, studying inhibitor of TGF‐β signaling pathway may provide new therapeutic strategies of those diseases. Pentachloropseudilin (PClP), a chlorinated phenylpyrrole compound isolated from Actinoplanes sp., and found in marine origin, was identified as an allosteric inhibitor of myosin family. However, how PClP influence TGF‐β signaling remains unknown. In this study, we found that PClP is a potent inhibitor of transforming growth factor‐beta signaling. PClP suppressed TGF‐β‐induced PAI‐1 reporter activation and Smad2/3 phosphorylation with a low IC50. PClP also inhibited TGF‐β‐induced epithelial–mesenchymal transition (EMT) and cell migration. Results from cell surface labeling and immunoblot analysis indicated that PClP suppressed TGF‐β response by promoting degradation of TGF‐β type II receptor (TβR‐II). The proposed mechanism is that PClP directs TβR‐II translocation to non‐raft fraction leading to accelerated internalisation and rapid degradation of TβR‐II.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Screening of in silico identified inhibitors of breast cancer resistance protein (BCRP) transporter in BCRP‐overexpressing MCF‐7 M100 cancer cells
Authors: Bequeaith Beau A. , Nanayakkara Amila K. , Vogel Pia D. , Wise John G. 
Published: June 2021

The overexpression of breast cancer resistance protein (BCRP) is associated with multidrug resistance (MDR) in specific cancers. BCRP is an ATP‐binding cassette (ABC) transporter that is also functionally important in the blood brain barrier. The ability of BCRP to export cancer chemotherapeutics to sub‐therapeutic concentrations renders chemotherapy treatment ineffective in cancers that overexpress BCRP. BCRP can also be problematic in its ability to inhibit the delivery of drugs to the brain. There are currently no clinically approved inhibitors of BCRP. The identification of inhibitors of BCRP that have the potential for clinical use would therefore greatly improve drug delivery systems to both cancers and the brain.Using computational approaches, we have identified a number of small, drug‐like molecules that have been predicted to inhibit BCRP. We report here the results of testing these small molecules in vitro in a BCRP‐overexpressing breast cancer cell line (MCF‐7 M100). The compound, Hoechst 33342, is known to be a transport substrate of BCRP, and was used here in assays that attempted to identify BCRP inhibition. Since Hoechst 33342 can be pumped out of the cells by BCRP, inhibition of BCRP leads to its intracellular accumulation. The BCRP activity assay used here takes advantage of the fact that accumulated Hoechst 33342 binds to DNA in the nucleus where it becomes highly fluorescent. The fluorescence of cells treated with putative inhibitors was compared with vehicle treated cells as an indication of BCRP inhibition. Nearly 100 experimental compounds were assayed at 10 μM in this study and compounds that exhibited three‐fold or greater accumulation over the vehicle treated control group were tested for inhibition of BCRP activity at lower concentrations. Five compounds have been identified so far that have demonstrated effective BCRP inhibition at nM concentrations. These compounds are currently being tested for the ability to reverse MDR in the BCRP‐overexpressing MCF‐7 M100 cell line.Support or Funding InformationThis work was supported by NIH NIGMS [R15GM09477102] to John G. Wise, SMU University Research Council, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, the Hamilton Undergraduate Research Scholars Program, and a private gift from Ms. Suzy Ruff of Dallas, Texas.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure‐based drug design targeting the malty sweet Mycobacterium tuberculosis GlgE
Authors: Petit Cecile , Kapil Sunayana , Veleti Sri Kumar , Lindenberger Jared , Sucheck Steven J. , Ronning Donald R. 
Published: June 2021

In 2016, 8.1 % of new cases (601,000 people) and 20 % of previously treated cases of Tuberculosis (TB) were diagnosed as Multiply Drug Resistant Tuberculosis (MDR‐TB) cases, amongst which 10 % are Totally Drug Resistant Tuberculosis (TDR‐TB) cases worldwide. In America, MDR‐TB new cases make 1.5 % of the cases among which none of the TDR‐TB cases were successfully treated in 2016. In addition to TB and HIV treatment being contraindicated, the rise of MDR‐TB and TDR‐TB emphasize the need for new drugs and drug targets. Identified in 2010, the maltosyltransferase GlgE is an interesting drug target in that it has no homologues in humans and its inhibition results in the death of the microorganism in 14 days. Based on the structure of GlgE's natural ligand, maltose‐1‐phosphate, and the known enzymatic mechanism, substrate and transition‐state analogs were designed as potential inhibitors of the enzyme and co‐crystallized with an MtbGlgE homolog. To improve the resolution of our X‐ray structures to more accurately analyze the interactions between the inhibitors and the residues in the active site, ScoGlgEI has been used as a surrogate. Sharing 53 % identity with MtbGlgE, we created a ScoGlgEI‐V279S variant as to obtain 100 % identity between the active sites of the two homologues. Based on the information obtained from X‐ray structures of ScoGlgEI‐V279S in complex with our initial inhibitor, 2,5‐dideoxy‐3‐O‐α‐D‐glucopyranosyl‐2,5‐imino‐D‐mannitol (DDGIM), a 2‐fold inhibition improvement has been made on our second‐generation inhibitors bringing the IC50 from 102 ± 8 μM to 45 ± 4 μM. Recently published, the crystal structures of ScoGlgEI‐V279S in complex with these early‐ and late‐dissociative transition‐state‐like inhibitors were solved at 2.5 Å and 3.2 Å showing that improvement was obtained through the addition of a phosphonate to our DDGIM inhibitor, resulting in an increased number of hydrogen bonded interactions between the enzyme and the inhibitors. To increase potency of our next generation inhibitor, additional interactions with the enzyme were sought outside the active site. The release of a structure of ScoGlgEI‐V279S in complex with its substrate brought more information to increase the number of interactions between ScoGlgEI‐V279S and the next generation of inhibitors. The details of the crystal structure of ScoGlgEI‐V279S in complex with our third‐generation inhibitor was obtained at 3.35 Å and the determined inhibitory activity will be discussed.Support or Funding InformationNIH grant AI105084This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.