bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biosynthesis of oxetanocin: are two cofactors better than one?
Authors: Bridwell‐Rabb Jennifer , Liu Hung‐wen , Zhong Aoshu , Drennan Catherine 
Published: June 2021

The biosynthesis of the natural product oxetanocin has been connected to a plasmid‐borne Bacillus megaterium gene cluster that contains two genes involved in oxetanocin production (oxsA and oxsB). Here, we present detailed structural and biochemical analysis that confirms the involvement of the cobalamin (Cbl) dependent S□adenosylmethionine (AdoMet) radical enzyme, OxsB, and an HD‐domain phosphohydrolase enzyme, OxsA, in oxetanocin production. These studies of OxsB provide a framework for understanding the coordination of AdoMet and Cbl in performing chemistry and reveal an HD‐domain phosphohydrolase enzyme with a restructured active site specific for a phosphorylated oxetanocin compound. Our crystallographic endeavors have allowed for elucidation of a biochemical scheme for the production of oxetanocin, expanded the catalytic repertoire of the HD‐domain phosphohydrolase enzyme superfamily, and provided the first structure of a Cbl‐dependent AdoMet radical enzyme.Support or Funding InformationSupport for this work came from National Institute of Health Grants F32‐GM108189 (J.B.‐R.) and GM035906 (H.‐w.L.), and the Welch Foundation grant F‐1511 (H.‐w.L.). C.L.D. is a Howard Hughes Medical Institute Investigator. This work is also based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P41 GM103403). The Pilatus 6M detector on 24‐ID‐C beam line is funded by a NIH‐ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE‐AC02‐06CH11357.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression, purification, and characterization of halophilic Pph_Pro1 protease isolated from Pseudoalteromonas phenolica
Authors: Choi Kwon‐Young , Kim Yun‐Gon , Yang Yung‐Hun 
Published: June 2021

In this study protease Pph_Pro1 from Pseudoalteromonas phenolica which was reported to have extracellular proteolytic activity and salt resistance properties was investigated for cloning, expression and purification. Through the optimization it was determined that when Pph_Pro1 was co‐expressed with pTf16 vector chaperone and grown with LB medium supplemented with CaCl3, optimum soluble recombinant expression was achieved. The Pph_Pro1 was purified using osmotic shock and immobilized metal‐affinity chromatography (IMAC) methods. The activity of the isolated Pph_Pro1 was determined as 0.44 U/mg against casein. Interestingly the Pph_Pro1 displayed halophilic, alkaliphilic, and unexpected thermostability properties. Furthermore, it was also resistant to a number of hydrophilic and hydrophobic organic solvents. The substrate specificity and kinetic values such as Km and Vmax were determined against casein, Bovine Serum Albumin (BSA) and algal waste protein, which indicated that the wild type proteases had a greater affinity for casein. Based on the outstanding characteristics of the wild type protease, obtained through experiments, it can find application in many biotechnological industries.Support or Funding InformationThis work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (NRF‐2015R1A2A2A04006014).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Radical SAM mechanism mediated by the Interferon‐Inducible Protein Viperin
Authors: Dumbrepatil Arti Baban , Malec Paige , Ghosh Soumi , Patel Ayesha , Kennedy Robert T. , Marsh E. Neil G. 
Published: June 2021

To survive infection the immune system deploys an arsenal of defensive measures to combat invading pathogens. The type I interferon (IFN) response presents an early host defense by generating an intracellular environment that restricts virus replication and signals the presence of a viral pathogen to the adaptive arm of the immune response. Viperin (virus inhibitory protein, endoplasmic reticulum‐associated, interferon‐inducible) is an interferon stimulated gene (ISG), that is induced by type I, II, and III IFNs, or after infection by a broad range of DNA and RNA viruses. Viperin contains an S‐adenosyl‐L‐methionine (SAM) domain and an N‐terminal amphipathic helix that contributes to its antiviral activity by localizing viperin to ER membranes or lipid droplets. Viperin has been found to require ER localization for full antiviral activity and mutation of the cysteine residues (CX3CX2C motif) ligating the Fe‐S cluster in the central radical SAM domain entirely abolished antiviral activity. Viperin induces a cascade of signaling events to mount an immune response against the viral replication; however, the exact molecular basis for viperin's cellular activity and the role of SAM‐dependent reactions in inhibiting viral synthesis remains unknown. Our studies suggest that the radical SAM activity of viperin is critical for activation of innate immune signaling. The enzyme localizes the E3 ubiquitin ligase TRAF6 and the interleukin 1 receptor‐associated kinase (IRAK1) to the endoplasmic reticulum and activates TRAF6 to catalyze polyubiquitylation of IRAK1, key step in the Toll‐like receptor‐7 (TLR) and TLR‐9 signaling pathways. Our data implicates the radical SAM activity of viperin is essential in this activation step. We found that in presence of IRAK1 and TRAF6 the reductive cleavage of SAM to form 5′‐deoxyadenosine is increased ~ 4‐fold. Thus, our data provides the first evidence that viperin's radical SAM activity is linked to activation of a ubiquitin ligase. This observation has implications for how the enzyme may exert its multifaceted roles in the cellular antiviral response.Support or Funding InformationThis work was supported in part by NIH grants GM 093088 to E.N.G.M. and DK 046960 to R.T.K.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthesis of 1,4‐diaminoanthraquinones for the purification of lactate dehydrogenase
Authors: Campbell Meagan Elizabeth , Mueller Jaime 
Published: June 2021

Cibacron blue 3G‐A is a dye commonly used for the purification of enzymes using affinity chromatography, often utilized in undergraduate biochemistry laboratory studies. Unfortunately, companies only have this product available intermittently, and when available it is sold at a fairly high price. Due to these issues, an alternative molecule that could be easily synthesized and would be similarly efficient at enzyme purification is desired. A distinct feature of Cibacron blue is its anthraquinone core. Anthraquinones are naturally‐occurring aromatic compounds commonly used in the manufacture of dyes. This synthesis utilizes a method of converting 1,4‐diaminoanthraquinones into diazonium salts to then allow the addition of various other functional groups, including anilines and phenols. After synthesis has been conducted, this small library of aminoanthraquinone derivatives will be tested for biological activity by examining spectrophotometric activity in the presence of lactate dehydrogenase (LDH). This will determine whether the products synthesized are efficient at binding to LDH and whether they can be successfully utilized for purification methods. This experiment details a simple, two‐step synthesis of various 1,4‐disubstituted aminoanthraquinone derivatives, which may be found to be useful alternatives to Cibacron blue. In addition, these products could potentially have other biological activity, due to the highly‐activated anthraquinone core.This 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 Proteomic Applications Using Anti‐DYKDDDDK Magnetic Agarose
Authors: Benton Betsy , Geddes Joanna , Vattem Krishna , Patel Bhavin , Kaboord Barbara 
Published: June 2021

Affinity purification, immunoprecipitation and mass spectrometry analysis are essential tools used in proteomic studies. A successful outcome is facilitated by high capacity supports, efficient sample preparation, and reproducibility provided by automation. We demonstrate that an anti‐DYKDDDDK magnetic agarose fulfills these criteria. The DYKDDDDK epitope tag is commonly used in protein expression systems as a short purification handle. Using an antibody against DYKDDDDK tag covalently attached to high binding capacity magnetic agarose, we demonstrate effective purification of recombinant proteins expressed in bacteria, the Invitrogen™ ExpiCHO Expression System and by a HeLa in vitro translation (cell‐free) system. Biological activity was demonstrated for ExpiCHO‐expressed DYKDDDDK‐Tumor necrosis factor alpha (TNFa) which was used to successfully stimulate cells and activate Nuclear Factor kappa B (NFkB). In addition, successful co‐immunoprecipitation of DYKDDDDK‐tagged BAD protein with 14‐3‐3 proteins was confirmed using an optimized sample preparation protocol in conjunction with the Thermo Scientific KingFisher Flex magnetic particle processor and mass spectrometry. In summary, anti‐DYKDDDDDK magnetic agarose can be used to effectively isolate, purify, and enrich functional protein targets in a variety of proteomic applications.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mass spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation
Authors: Levy Ezra , Budnik Bogdan , Slavov Nikolai 
Published: June 2021

Cellular heterogeneity is important to biological processes, including cancer and development. However, proteome heterogeneity is largely unexplored because of the limitations of existing methods for quantifying protein levels in single cells. To alleviate these limitations, we developed Single Cell ProtEomics by Mass Spectrometry (SCoPE‐MS), and validated its ability to identify distinct human cancer cell types based on their proteomes. We used SCoPE‐MS to quantify over a thousand proteins in differentiating mouse embryonic stem (ES) cells. The single‐cell proteomes enabled us to deconstruct cell populations and infer protein abundance relationships. Comparison between single‐cell proteomes and transcriptomes indicated coordinated mRNA and protein covariation. Yet many genes exhibited functionally concerted and distinct regulatory patterns at the mRNA and the protein levels, suggesting that post‐transcriptional regulatory mechanisms contribute to proteome remodeling during lineage specification, especially for developmental genes. SCoPE‐MS is broadly applicable to measuring proteome configurations of single cells and linking them to functional phenotypes, such as cell type and differentiation potentials.Support or Funding InformationNortheastern UniversityNIH New Innovator Award DP2GM123497This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cell surface GRP78 activation by anti‐GRP78 autoantibodies in relation to prostate tumour growth via tissue factor activation.
Authors: Al‐Hashimi Ali , Shayegan Bobby , Austin Richard 
Published: June 2021

BackgroundProstate cancer (PC) presentation is associated with the production of anti‐GRP78 autoantibodies that enhance tissue factor (TF) expression and procoagulant activity (PCA). We and others observed that GRP78 is expressed on the surface of PC cells where it functions as a signaling receptor to promote cell proliferation and survival. Normally, GRP78 is an endoplasmic reticulum resident chaperon that ensure the proper folding of newly synthesized polypeptides. We report that binding of these autoantibodies to cell surface GRP78 PC progression in mice, mediated by TF. Further, this observed increase in tumour growth can be reversed with the use of heparin or low molecular weight heparin molecules. We hypothesize that the disruption of the autoantibody/cell surface GRP78 complex represent a viable surrogate treatment of PC.MethodsWild type, TF knockdown DU145 cells, and NOD/SCID mouse model system was used to investigate the effect of anti‐GRP78 autoantibodies on tumor growth. Protein expression was determined using western blotting and qRT‐PCR. TF activity was determined using the TF PCA continuous assay. Blood samples from patients diagnosed with PC were obtained from the Ontario Tumour Bank and St. Joseph's Hamilton.Results

‐Anti‐GRP78 autoantibodies correlate with PC stage: PC patients demonstrated high levels of anti‐GRP78 autoantibodies (60–100 μg/ml, pre‐prostatectomy stage) vs. healthy individuals (5–10 μg/ml). These titers were significantly reduced 24‐weeks post‐prostatectomy.
‐Anti‐GRP78 autoantibodies drive tumour progression: We show here that anti‐GRP78 autoantibodies activate the unfolded protein response, a pro‐survival cellular pathway in cancer cells. Furthermore, these autoantibodies were shown to accelerate tumor growth in a NOD/SCID mouse model.
‐Heparin and low molecular weight heparin abolished the effect of anti‐GRP78 autoantiboides on PC progression. In vitro: We show that heparin and low molecular weight heparin inhibit the binding of anti‐GRP78 autoantibodies. In vivo: a low molecular weight heparin was used as an intervention treatment for anti‐GRP78 autoantibodies and was shown to reverse the effect of anti‐GRP78 autoantibodies on increased tumour growth.
ConclusionsWe have identified the function of an agent in patients' blood, anti‐GRP78 autoantibodies, that correlate with PC stage in patients and increase TF PCA and promote PC progression in mice. The effect of this autoantibody can be reversed using heparin, thus, this acts as a new potential therapeutic target for PC.Support or Funding InformationThis work was supported in part by Prostate Cancer Canada (PCC) Discovery Grant (Discovery Grants # 2010‐590, and D2017‐1949), and McMaster Surgical Associate Grant 176725.
27‐hydroxycholesterol decreases cell proliferation in colon cancer cells
Authors: Warns Jessica A. , Freking Natalie , Ghribi Othman 
Published: June 2021

In the United States, Colorectal Cancer is the third most diagnosed cancer, affecting 1 out of approximately 22 people in their lifetime. A major molecule involved in cancer progression is the AKT signaling pathway, which regulates cell cycle progression, protein synthesis, and cellular survival. Several cholesterol metabolites, including 27‐hydroxycholesterol (27‐OHC) have been shown to be involved in breast and prostate cancers. However, the role of 27‐OHC in colon cancer has yet to be determined. The goal of our study is to investigate the potential role of 27‐OHC in colon cancer progression. To study the effects of 27‐OHC, SW620 and Caco2 colon cancer cells were treated with 0.5 μM to 300 μM of 27‐OHC for 24 hours and MTT proliferation assay, LDH cytotoxicity, TUNEL assay, and western blotting was performed. Additionally, cellular migration was measured with a scratch assay. We found a significant decrease in cellular proliferation at supraphysiological concentrations of 27‐OHC (10 μM to 300 μM) with no significant cellular cytotoxicity or apoptotic cell death. Western blotting showed a significant decrease in p‐AKT expression with 10 μM of 27‐OHC in Caco2 cells. In both cell lines there was a significant increase in cellular migration. This data demonstrates that 27‐OHC increases cellular migration through a loss of cellular proliferation. The decrease in cellular proliferation is dependent on p‐AKT in Caco2 cells and an AKT‐independent pathway in SW620 cells, suggesting that 27‐OHC has differential effects on different colon cancer cells.Support or Funding InformationR01AG0145264 awarded to OGThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanosensitivity Analysis of Breast Cancer Tumor Cells from Needle Biopsy
Authors: Bedoya Santiago Acero , Ghosh Deepraj , Dawson Michelle 
Published: June 2021

Tumor stiffness has been associated with malignancy and increased risk for metastasis. Extensive research has been done investigating breast cancer cell lines' responsiveness to surfaces of varying rigidities as well as examining the biophysical properties of breast cancer tumor samples. However, there is a critical gap regarding the relationship between cells' mechanosensitivity in conjunction to biophysical properties of their extracellular matrix environment. To explore this relationship, we will analyze single‐cell mechanosensitivity in comparison to tumor rigidity via shearwave ultrasound elastogrophy (SWE). Given the putative affiliation, we hypothesize that cells expressing invasive mechanosensitivity profiles will correlate with stiffer tumor regions. Using collagen gels containing different cell types, we derived biopsy‐sized samples allowing us to optimize single‐cell mechanosensitivity analysis. Cells were stained using different dyes corresponding to invasiveness. Subsequently, we analyzed their morphology. Morphological identification within organoid environments would allow for single‐cell analysis without the aggression of tissue digestion, though preliminary results suggest high heterogeneity may not allow for confident cell identification solely on morphology. Thus, inquisition into cell viability and integrity was explored by analyzing the effects of tissue digestion with HyQtase on single‐cells. Cell count and live‐dead stain via flow cytometry allowed for analysis of single‐cell viability. Lastly, cell integrity was evaluated by a 2D adhesion assay of isolated cells. The live/dead stain revealed that digestion resulted in isolation of approximately 10% of the original 500,000 cell population with 90–97% of the isolated population being live‐cells (invasive and non‐invasive respectively). Furthermore, the adhesion assay showed that these isolated single cells retained the ability to adhere to new surfaces, with no difference between the invasive and non‐invasive cell types. These results show that cells are able to retain mechanosensitive properties following enzymatic digestion. However, they also suggest our digestion procedure is not aggressive enough to isolate invasive subpopulations that are more strongly imbedded in the original tissues. Development of these novel techniques will allow for accurate and confident analysis of precious human biopsy samples. Insight into the relationship between single‐cell mechanosensitivity and tumor biophysical properties could elucidate pathways for metastasis inhibition and prevention.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Combined Effect of Canagliflozin and Metformin in Human Prostate Cancer Cells
Authors: Ware Kierra , Stewart LaMonica 
Published: June 2021

BackgroundThere is still a need to identify effective treatments for castration‐resistant prostate cancers. Recent studies have shown that two drugs used to treat type 2 diabetes, metformin and canagliflozin, may be effective treatments for castration‐resistant prostate cancer. Both drugs have individually been shown to inhibit proliferation of 22Rv1 and PC3 prostate cancer cell lines. However, it is not known if combined treatment with metformin and canagliflozin suppresses the growth of prostate cancer cells better than each drug alone. The goal of this study was to determine the effectiveness of the combination of canagliflozin and metformin as an alternative treatment for prostate cancer.MethodsPresto Blue Assays were used to assess the effect of metformin and canagliflozin on proliferation of the 22Rv1 and PC3 prostate cancer cell lines. Western blot analysis was also performed to determine the effect of metformin and canagliflozin (both individually and combined) on androgen receptor (AR) protein levels. Western blots were stripped and re‐probed for the protein actin to confirm that gels were evenly loaded.ResultsCanagliflozin at a concentration of 30 uM produced a significant decrease in the proliferation of PC3 and 22Rv1 cells. While metformin showed no significant decrease in proliferation of PC3 cells, metformin (5 mM) did significantly decrease 22Rv1 cell proliferation. The combination of canagliflozin and metformin showed a significant decrease in both PC3 and 22Rv1 cells. However, the effect of the combination was no greater than canagliflozin alone. AR protein levels were reduced by canagliflozin, metformin, and the combination of both drugs. Moreover, the combination of metformin and canagliflozin was the most effective in reducing AR protein levels.ConclusionThe combination of canagliflozin and metformin was equally effective as canagliflozin alone at reducing prostate cancer cell proliferation. However, this drug combination was more effective at suppressing AR protein levels within castration‐resistant prostate cancer cells. Taken together, these data suggest that combination treatments involving metformin and canagliflozin could be used to decrease tumor growth and AR expression in prostate cancer patients who have developed castration‐resistant prostate cancer.Support or Funding InformationSupported by NIH grants MD007593 and T34GM007663This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A vitamin B12 receptor serves a role in membrane stability of Caulobacter crescentus
Authors: Barraza Daniela , Strebe Sierra , Menikpurage Inoka , Melendez Ady , Mera Paola E. 
Published: June 2021

This project focuses on studying the function of a highly expressed receptor in bacteria. In E. coli, the BtuB transmembrane receptor is involved in the active transport of vitamin B12. The role of BtuB in our model organism C. crescentus is currently unknown. Recently, this protein was shown to be highly expressed (levels comparable to ribosomal proteins) throughout the cell cycle of C. crescentus (Schrader et al 2016). This is intriguing since C. crescentus is a fresh water bacterium that is not likely to be exposed to vitamin B12. We hypothesize that this membrane receptor serves to stabilize the outer membrane of C. crescentus. We began by constructing a btuB deletion mutant and collecting data regarding morphology, growth rate, motility, and viability characteristics as compared to wild type. We showed that BtuB in C. crescentus plays a role in vitamin B12 transport when nutrient availability is limited. Using Transmission Electron Microscopy we observed the mutant to have a potential defect in membrane stability when the cells are growing at high doubling rates. In conclusion, our data suggests that this membrane receptor is playing a role in membrane integrity aside from vitamin B12 transport. Currently, we are in the process of testing membrane stability characteristics of the ΔbtuB mutant. In addition, we are employing a combination of genetics, biochemistry, and microscopy techniques to see if BtuB's role in membrane stability is dependent on vitamin B12 transport.Support or Funding InformationFunding by Maximizing Access to Research Careers (MARC), grant number: GM07667‐39This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.