bluuebunny/crossref_metadata_2025 huggingface dataset:
Genesis of antibiotic resistance (AR) III: trifling risk of AR pathogens induced infectious diseases from regulated concentrated animal feeding operations (986.3)
Authors: Kannan Subburaj , Hernandez Lizeth , Herrera Ana , Jimenez Brenda , Miller Monica , Perales Patricia , Reyna Cristal , Subburaj Prabavathy
Published: June 2021

Concentrated Animal Feeding Operations (CAFOs) are sites where animals are housed and raised in a confined environment. CAFOs assemble animals that are fed and cared for by professional care givers referred to as “Cowboys” on a limited space in a rustic landscape. The operation is monitored and meets the standards set forth by The United states Environmental Protection Agency (http://www.epa.gov/region7/water/cafo/). In southwestern Texas, cattle are imported from Mexico, and subjected to the health inspection by The United States ‐ Mexico border USDA‐APHIS‐VS stations. Cattle undergo rigorous inspections for Tuberculosis, tick‐borne infections, and respiratory illness, along with prior health records followed by consignment to CAFOs (Feed Lots). While in Feed lots, the health and well being of the cattle is monitored by the house staff and also by USDA veterinarian. The amount of “Antibiotics” supplemented in the cattle feed is regulated by the EPA standards and followed through by in house nutritionist. MICRO BEEF TECHNOLOGIES, Ltd (http://www.microbeef.com/about_micro/index.html ) provides an excellent tracking system to maintain comprehensive details on individual animals regarding their health and nutrition. Taken together with an exceptional monitoring programs by The US‐EPA, USDA‐APHIS‐VS, and strict industry standards upheld by The Texas Cattle Feeders Association, the CAFOs pose minimal risk of rendering AR pathogen affected infectious diseases. (Acknowledgement: Dr. Darrell Haney, USDA‐APHIS‐VS, (830) 752‐1785, Eagle Pass, Texas; Mr. Luis De La Cruz, Runnells‐Peters Feed Yard LLC, 1444 Fm 1665, Quemado, TX 78877‐7790; Information Resources from Texas Cattle Feeders Association, http://www.tcfa.org/SiteMap.html).

Grant Funding Source: Professional Development Funds ‐ SWTJC, Eagle Pass, Texas 78852
Evidence for an interdomain flavin binding site in Steap (six‐transmembrane epithelial antigen of the prostate) family metalloreductases associated with iron homeostasis and insulin resistance (996.4)
Authors: Kleven Mark , Gauss George , Fleming Mark , Lawrence C.
Published: June 2021

Steap family enzymes are integral membrane NADPH oxidases capable of reducing both Fe3+ and Cu2+ at the cell surface. While Steap3 has previously been identified as the ferrireductase in the transferrin cycle and Steap4 is linked to obesity associated insulin resistance, cellular roles and chemical underpinnings of the family remain ill‐defined. Here we report in‐depth biochemical and structural characterization of Steap3 and Steap4. Consistent with both cell surface and endosomal activities, they show significant metalloreductase activity over a broad pH range, and recognize both Fe3+ and Cu2+ at physiologically relevant concentrations. Structure‐function studies also reveal the absence of a high affinity flavin binding site in the isolated N‐terminal oxidoreductase domains. Full‐length proteins, however, show high affinity binding sites for FAD. This suggests that Steap family members may also function in copper transport, and that an interdomain flavin binding facilitates electron transfer between NADPH of the oxidoreductase domain, and a heme cofactor in the transmembrane domain. In addition, bimolecular fluorescence complementation (BiFC) indicates they are active as homodimers.

Grant Funding Source: Supported by the National Institutes of Health, Grant #: GM084326
Cellular zinc levels are influenced by the interaction between the mucolipidosis IV‐associated TRPML1 protein and TMEM163 protein (996.5)
Authors: Silva Joshua , Basilio Luigi , Hart Thomas , Tringali Jonathan , Cuajungco Math
Published: June 2021

Dysfunctional Mucolipin‐1 (TRPML1) ion channel protein causes the human lysosomal storage disorder Mucolipidosis type IV (MLIV). Cells affected by MLIV have significantly higher intracellular zinc levels compared to normal cells; however, the mechanism of zinc elevation remains unknown. Using a membrane‐based yeast two‐hybrid technique, we identified a putative zinc‐binding protein called transmembrane‐163 protein (TMEM163) as an interacting partner for TRPML1. We confirmed interaction by co‐immunoprecipitation and mass spectrometry. We hypothesized that the interaction between TMEM163 and TRPML1 plays a role in regulating TMEM163, and thus intracellular zinc. RNA interference targeting both TMEM163 and TRPML1 in SH‐SY5Y neuroblastoma cells resulted in significant increase of intracellular zinc when both proteins were knocked down. To determine how interaction of TRPML1 and TMEM163 could be responsible in regulation of intracellular zinc, we co‐expressed and analyzed expression of tagged constructs of both proteins using human embryonic kidney (HEK)‐293 cells. Evidence from cell surface biotinylation showed that co‐expressed TRPML1 resulted in reduced TMEM163 localized within the plasma membrane of HEK‐293 cells. This result possibly explains the reduction of cellular zinc flux when TRPML1 is co‐expressed with TMEM163. Overall, our data suggest that both TRPML1 and TMEM163 proteins play a role in intracellular zinc regulation. Thus, the loss of TRPML1 protein function and/or the reduction of TMEM163 protein could result in zinc dyshomeostasis in MLIV.

Grant Funding Source: Supported by NIH R15‐NS070774 and CSUF Intramural Grants
Cross‐linking study of membrane subunits K, L, M and N in E. coli complex I (997.10)
Authors: Vik Steven , Zhu Shaotong , Tursun Ablat
Published: June 2021

Complex I is an entry point of the electron transport chain and plays a major role in proton gradient generation. Oxidation of NADH, reduction of ubiquinone, and proton translocation are three essential functions of Complex I. Subunits L, M, and N, the three largest subunits in the membrane domain, are homologous to components of multi‐subunit Na+/H+ antiporters, so they are implicated in proton pumping. Based on available evidence it is believed that a redox‐linked conformational change is necessary for proton pumping. According to recent crystal structures, subunit L contains an unusual helix HL extending laterally along the membrane surface from subunit L, past subunit M, and to subunits N and K. In this study, cross‐linking of engineered cysteine residues using disulfide formation and bi‐functional reagents was carried out to detect regions of the proton transporter that have restricted motion. Fourteen cysteine pairs between helix HL and membrane subunits L, M, N and K were cross‐linked and none showed no significant loss of enzyme activity or proton pumping These results suggest that there is not significant movement of the lateral helix. Further cross‐linking studies were done between pairs of cysteine residues within subunit N. The results of those studies suggested possible proton pathways.

Grant Funding Source: NIH 1R15GM099014
Identification of trafficking motifs in the C‐terminus of the cystine/gluatamate exchanger, System xc‐ (997.3)
Authors: Ladd Nicole , Unterbrink Elizabeth , Georges Anne , Lang Sara , Chase Leah
Published: June 2021

System xc‐ is a heterodimeric plasma membrane transporter, comprised of the proteins xCT and CD98, which facilitates the stoichiometric exchange of extracellular cystine for intracellular glutamate. System xc‐ serves to protect the cell from oxidative stress, and brief exposure of cells to oxidative agents leads to an increase in trafficking of xCT to the plasma membrane. However, little is known about the mechanisms which govern the constitutive and regulated trafficking of xCT. In the present study, we sought to identify trafficking motifs within the C‐terminus of xCT that are important in regulating the delivery and internalization of xCT from the membrane. We used a mutagenesis approach combined with cell surface biotinylation assays to identify regions of the C‐terminus that are involved in the constitutive trafficking of xCT. Specifically, using site‐directed mutagenesis, we identified a tyrosine based motif between positions 464 ‐486 containing the sequence YYFII that contributes to the constitutive trafficking of xCT to the plasma membrane. In addition, we created three truncation mutants at positions 469, 484, and 496 of xCT, and found that an endocytic motif(s) must also exist between position 469 and 484. While there are no canonical endocytic motifs within this region, there is a putative ubiquitination site. Since we have recently demonstrated that xCT is ubiquitinated in Cos‐7 cells and ubiquitination of membrane proteins is known to regulate cell surface expression, we are currently using site‐directed mutatgenesis to test the hypothesis that ubiquitination of xCT in this region leads to the increased rate of internalization of xCT. This research was supported in part by NSF‐RUI #0848564.

Grant Funding Source: NSF‐RUI #0848564
Single molecule studies of the E.coli protein export system (997.9)
Authors: Sanganna Gari Raghavendar Reddy , Frey Nathan , Mao Chunfeng , Randall Linda , King Gavin
Published: June 2021

The general secretory or Sec system is the major route of export for proteins from the cytosol of Escherichia coli. The pathway through the membrane is provided by the translocon SecYEG. At the membrane, ATPase SecA binds SecYEG and drives protein translocation. In this work we shed light on Sec system using atomic force microscopy (AFM), a single molecule technique that is well suited for studying membrane proteins in near native conditions. AFM images provided direct visualizations of the dynamic loops of SecYEG which orchestrate critical translocon functions as well as determination of the oligomeric state of SecYEG in lipid bilayers. Additionally, we studied structure‐function relationships of SecA‐SecYEG complexes. Our results show that SecA binds SecYEG in liposomes in two distinct modes. When SecA was added extraneously to liposomes containing SecYEG, the proteins showed a wide distribution of heights with no prominent peaks, indicating that SecA populates multiple binding states. In contrast to this broad distribution of heights, the sample of proteoliposomes in which SecY and SecA were co‐assembled had a predominant species centered at 40 Å in height above the lipid bilayer. Because these proteoliposomes contain approximately sixfold more active translocons than do the proteoliposomes to which SecA was added after assembly, we conclude that this structural state represents SecA bound to translocons that have been rendered active. Taken together this work provides new functional and structural insights into the Sec system. More generally, the assays developed here are adaptable to other membrane protein systems.

Grant Funding Source: NSF CAREER Award , Burroughs Wellcome Fund Career Award, NIH
Phosphatidylinositol‐4‐phosphate synthesis and turnover spans multiple membrane compartments (999.2)
Authors: Hammond Gerry , Machner Matthias , Balla Tamas
Published: June 2021

Organelle identity may be assisted by a phosphoinositide (PI) code, wherein specific PI lipids act as molecular “signposts”, ensuring exclusive recruitment of binding proteins to the correct membrane compartment. One example is PtdIns4P, whose known effector proteins are recruited to the Golgi. However, the PI 4‐kinases (PI4K) that make PtdIns4P belie such a restricted function, since they act at the Golgi, plasma membrane (PM) and in lysosomal traffic. To reconcile this contradiction, we re‐examined the distribution of PtdIns4P with a novel biosensor derived from the P4M domain of L. pneumophila SidM. P4M localizes to the Golgi, PM and dynamic Rab7‐positive late endosomes/lysosomes. Chemically‐induced recruitment of PI phosphatases to these compartments demonstrates PtdIns4P is the lipid bound in each case. Whereas the bulk of another PI lipid, PtdIns(4,5)P2 is localized at the PM, it could be produced in the other PtdIns4P‐containing compartments by recruitment of a PtdIns4P 5‐kinase. P4M can also detect ectopically produced PtdIns4P on ER‐derived membranes. We show that each of the four PI4Ks overlap with pools of PtdIns4P, and the ER/Golgi localized PtdIns4P phosphatase, Sacm1l, hydrolyses both Golgi and PM pools. We conclude that PtdIns4P has a much more complex function than as a simple molecular signpost for the Golgi, and likely functions through several as yet unappreciated protein interactions.

Grant Funding Source: Intramural Research Program of the Eunice Kennedy Shriver NICHD, NIH
Discovery of a lipid deacetylase (AADACL1) as a novel regulator of platelet activation (999.4)
Authors: Holly Stephen , Chang Jae , Li Weiwei , Niessen Sherry , Phillips Ryan , Piatt Raymond , Black Justin , Smith Matthew , Boulaftali Yacine , Weyrich Andrew , Bergmeier Wolfgang , Cravatt Benjamin , Parise Leslie
Published: June 2021

Hyperactive platelets can trigger thrombotic episodes including myocardial and cerebral infarction, whereas insufficient platelet reactivity can lead to spontaneous bleeding. To better understand these essential vascular processes, we screened human platelets with a carbamate library designed to interrogate the serine hydrolase proteome coupled with competitive activity‐based protein profiling (ABPP) to map the targets of active carbamates. Using this strategy, we identified a novel lipid hydrolase in human platelets named arylacetamide deacetylase‐like 1 (AADACL1) and delineated a critical role for this enzyme in response to multiple agonists, especially collagen. We showed that AADACL1 regulates lipid metabolism, RAP1 and PKC activation, fibrinogen binding, platelet aggregation and thrombus formation in an ex vivo model of collagen‐dependent thrombosis. Preliminary data also suggest that AADACL1 contributes to platelet activation in vivo, since rats injected with a selective AADACL1 inhibitor show reduced platelet aggregation in whole blood. While these data clearly implicate AADACL1 in platelet biology, the physiological role of AADACL1 is currently unknown, so future studies are needed to evaluate the scope of AADACL1 action in the vasculature and determine its value as a potential anti‐thrombotic target.

Grant Funding Source: AHA and the NIHLBI
P‐glycoprotein ‐ an ABC Transporter: Toxin Removal to Chemotherapy Interference (LB109)
Authors: Adamson Bennett , Pham Tiffany , Kronenfeld Jason , Reilly Bianca , Crowther Meghan , Fraijo Savanah , Cherrington Emily , Futch Liane , Cherrington Nathan
Published: June 2021

Cancer claimed 7.6 million lives worldwide in 2013. Despite improvements in chemotherapy, most relapsed cancers become drug resistant due to an ATP‐binding cassette transporter, P‐glycoprotein (P‐gp). P‐gp is a gatekeeper in multiple tissues, controlling exposure to drugs and toxins by pumping them out of the cell. This homodimeric protein of 1,280 amino acids is located on the apical surface and extrudes lipid soluble molecules. Pharmaceutical companies must determine if their drugs are P‐gp substrates before proceeding to clinical trials, as the amount of drug entering the body may vary widely due to P‐gp function. P‐gp may reduce the amount of drugs reaching circulation by transporting drugs back into the digestive tract lumen. Many chemicals act as modulators of P‐gp’s function; for example, the flavonoid naringin in grapefruit juice interacts with P‐gp and may significantly increase the uptake of some statin drugs. The active sites and conformational changes of P‐gp have not been elucidated, as it has been difficult to crystallize P‐gp with its substrates. P‐gp’s complex structure and multiple binding sites allow for interaction with many drug molecules, which enter P‐gp’s binding pocket via pairs of alpha‐helices on opposite sides of P‐gp. The Ironwood Ridge High School SMART team (Students Modeling A Research Topic) modeled P‐gp using 3D printing technology.

Grant Funding Source: Supported by a grant from NIH‐SEPA and NIH‐CTSA.
Mutations altering the interplay between GkDnaC helicase and DNA reveal an insight into helicase unwinding (LB125)
Authors: Hsiao Chwan‐Deng , Sun Yuh‐Ju , Li Hung‐Wen
Published: June 2021

DNA helicase is a motor protein that unwinds and separates duplex DNA using energy derived from the hydrolysis of nucleoside triphosphates (NTPs). In different organisms, helicase has a number of different mechanisms for unwinding activity. The studies of the helicase activity in complex with individual and sets of accessory proteins will elucidate the complexity of the regulatory network within the whole replisome machinery. In this study, we use the replicative helicase of Geobacillus kaustophilus, a gram‐positive bacterium, as a model system (GkDnaC). Based on our previous crystal structure of the GkDnaC‐ssDNA complex, we identified several residues involved in ssDNA binding. The locations of these key residues imply that these ssDNA‐interacting sites correlate with helicase translocation. To investigate the nature of unwinding by hexameric GkDnaC helicase (superfamilies IV), we studied how the activity of GkDnaC is modulated by specific mutations and by accessory proteins. Our results imply that a partial loss in the interaction of helicase with ssDNA leads to an enhancement in helicase activity, while their ATPase activities remain unchanged. Based on kinetic evidences from single‐molecule tethered particle motion (smTPM) experiments, the unwinding rates of these GkDnaC mutants were consistently faster than that of wild‐type enzyme. In addition, we characterized the role of accessory proteins of GkDnaC helicase, showing that the interaction with accessory proteins alters both the ATPase and the unwinding velocity of the helicase.
The discovery of a lactone isomerase involved in pectin degradation (LB137)
Authors: Bouvier Jason , Groninger‐Poe Fiona , Vetting Matthew , Almo Steven , Gerlt John
Published: June 2021

Pectin, an abundant plant cell wall polymer, is being investigated as a possible raw material for the production of fuel and chemical products. To this end, an understanding of the steps to degrade pectin is useful. Agrobacterium tumefaciens strain C58 can use the primary constituent monomer of pectin, D‐galacturonate, as a sole carbon source and converts it to α‐ketoglutarate through a five step pathway in which the first step is oxidation of D‐galacturonate to D‐galactaro‐1,5‐lactone. We became interested in this pathway because a member of the functionally diverse enolase superfamily, designated Gci, catalyzes the third step in the pathway, and our lab has studied the enolase superfamily for over 20 years. We have identified an enzyme, D‐galactarolactone isomerase (GLI), adjacent to Gci in Agrobacterium’s genome, that catalyzes the second step in the pathway: the isomerization of D‐galactaro‐1,5‐lactone to D‐galactaro‐1,4‐lactone. The uncatalyzed isomerization reaction is “fast” and had been presumed to occur without the need of a catalyst. To characterize the enzyme‐catalyzed isomerization, we used 1H NMR to identify the activity, polarimetry to determine the kinetic constants (kcat = 440 s‐1, kcat/Km = 8.3 x 104 M‐1 s‐1, rate enhancement = 6.8 x 105), x‐ray crystallography to determine its structure, and gene expression analysis to connect the in vitro activity of the enzyme to an in vivo function. GLI is a member of the functionally diverse amidohydrolase superfamily and a homologue of LigI that catalyzes the hydrolysis of 2‐pyrone‐4,6‐dicarboxylate in lignin degradation. GLI’s ability to catalyze lactone isomerization instead of hydrolysis can be explained by the absence of the general basic catalysis used by LigI. This work identified the first example of lactone isomerization in the amidohydrolase superfamily and a missing enzyme in Agrobacterium’s pathway to catabolize pectin.

Grant Funding Source: Supported by National Institutes of Health Grants P01GM071790 and U54GM093342 to J.A.G.
Structure‐function analysis of the exoribonuclease domain of arenaviral nucleoproteins in mediating host immune evasion (LB138)
Authors: Ly Hinh , Liang Yuying
Published: June 2021

Several pathogenic arenaviruses, including Lassa virus, cause hemorrhagic fever (HF) infections that can result in significant morbidity and mortality in humans. A hallmark of severe HF is the high levels of viremia coupled with generalized immune suppression of the hosts, the mechanisms for which are unknown. Recent studies in our laboratory have revealed several potential mechanisms that arenaviruses use to cause virulent infection [McLay, et al., JVI 2013, Jiang, et al., JBC 2013; Kumar, et al., Virol 2012; Wang, et al., JVI 2012; Qi, et al., Nature 2010; Lan, et al., JVI 2009, Arch Virol 2008). A unique feature of lethal Lassa (LASV) arenaviral infection is that it is often associated with a generalized immune suppression of the infected hosts, the exact mechanism of which is unclear but is thought to involve the ability of the viral nucleoprotein (NP) to suppress the induction of type I interferons (IFN). We have recently determined the first crystal structure for LASV NP at 1.80 Angstrom resolution (Qi, et al., Nature 2010), which has unexpectedly revealed its unique mechanisms to suppress interferon‐beta production. Our structurally directed functional assays (using both biochemical and cell‐ and virus‐based assays) have demonstrated that the C‐terminal domain of LASV NP contains exoribonuclease enzymatic activity that is required for suppressing IFN‐beta induction by degrading immune stimulatory RNAs. We have also recently produced evidence to suggest that this is a common mechanism used by all known arenaviruses (Jiang, et al., JBC 2013). Novel insights learned from these studies can be exploited for the development of novel therapeutics and vaccines against deadly HF infections as recently reviewed by us (McLay, et al., Antiviral Research 2012, J Gen Virol 2014).

Grant Funding Source: NIH R01 grants AI083409 to Y.L. and AI093580 to H.L.
Acute impairment of early T cell activation following impediment of mitochondrial electron transport (LB166)
Authors: Hwang Inkyu
Published: June 2021

T cells absorb nanometric membrane vesicles, derived from antigen presenting cells, via dual receptor/ligand interactions of T cell receptor (TCR) with cognate peptide/major histocompatibility complex (MHC) complex plus lymphocyte function‐associated antigen 1 (LFA‐1) with intercellular adhesion molecule 1. Our studies have shown that TCR‐mediated signaling is also critically involved in the vesicle absorption, which is likely for the activation of LFA‐1. In an effort to systematically investigate the signaling process involved in the vesicle absorption process, we exploited chemical biology tools and screened chemical libraries to identify small molecules to interfere with the absorption process. Interestingly, it was found in the course of the study that brief treatments (1 hour) with various mitochondrial antagonists, including complex I, II, III, IV and V inhibitors,, resulted in ubiquitous inhibition of the vesicle absorption at concentrations not compromising viability of T cells. Further studies revealed that the mitochondrial drug treatments caused impairment of specific membrane‐proximal TCR signaling event(s). Thus, the activation of Akt and PLC‐γ1 and entry of extracellular Ca2+ following TCR triggering were severely compromised, while polymerization of monomeric actins immediately monitored upon triggering of TCRs appeared to progress normally even after the treatments. Dynamic F‐actin rearrangement concurring with the vesicle absorption was, however, also found to be impeded by the drug treatments, implying that the inhibition by the drug treatments of downstream signaling events at least in part resulted from the impairment of directional relocation of signaling and cell surface molecules accompanying with actin rearrangement.

Grant Funding Source: Supported by Chungnam National University Grant (2013‐1892)
Regulation of mitochondrial functions by SIRT5 and non‐canonical post‐translational modifications (LB172)
Authors: Lombard David , Park Jeongsoon
Published: June 2021

Protein function is regulated by diverse post‐translational modifications. The mitochondrial sirtuin SIRT5 removes malonyl and succinyl moieties from target lysines. The spectrum of protein substrates subject to these modifications is unknown. We recently reported the first systematic profiling of the mammalian succinylome, identifying 2565 succinylation sites on 779 proteins. Our analysis revealed potential impacts of lysine succinylation on amino acid degradation, the TCA cycle, and fatty acid metabolism. Lysine succinylation was also present on cytosolic and nuclear proteins; indeed, we found that a substantial fraction of SIRT5 was extra‐mitochondrial. SIRT5 repressed biochemical activity of, and cellular respiration through, two protein complexes identified in our analysis, Pyruvate Dehydrogenase Complex (PDC) and Succinate Dehydrogenase (SDH). We will discuss our recent progress on elucidating further the biology of SIRT5 using enzymatic, respiration, and metabolomic flux assays.

Grant Funding Source: Supported by NIH awards R01GM101171 and R21CA177925 (D.L.).
Proteomic analysis of hyaluronate‐regulated proteins involved in chondroprotective effect under oxidative stress (LB177)
Authors: Yu Chia‐Jung , Ko Chun‐Jung , Hsieh Chang‐Hsun , Chien Chiang‐Ting , Huang Lien‐Hung , Lee Chien‐Wei , Jiang Ching‐Chuan
Published: June 2021

Osteoarthritis (OA), the most common type of arthritis, is a degenerative joint disease. Oxidative stress is well known to play important roles in cartilage degradation and pathogenesis of OA. The intra‐articular injection of hyaluronic acid (IAHA) is accepted as an effective clinical therapy for OA, but we do not yet fully understand the mechanisms underlying the effects of HA on OA chondrocytes under oxidative stress. Here, we show for the first time that IAHA significantly reduces the synovial fluid levels of hydrogen peroxide (H2O2) and superoxide (O2−) in patients with knee OA. We also demonstrate that HA suppresses H2O2‐induced cell death in human OA chondrocytes. Proteomic approaches (2‐DE combined with mass spectrometry) allowed us to identify 13 protein spots corresponding to 12 non‐redundant proteins as HA‐regulated proteins in OA chondrocytes under oxidative stress. The expression levels of three putative HA‐regulated proteins (TALDO, ANXA1 and EF2) in control, H2O2‐, HA‐ and HA/H2O2‐treated OA chondrocytes were verified by Western blotting and the results indeed support the notion that HA acts in anti‐oxidation, anti‐apoptosis, and the promotion of cell survival. Our results collectively demonstrate the utility of proteomic approaches and provide new insights into the chondroprotective effects of HA on OA.

Grant Funding Source: Supported by National Science Council, R.O.C.
A dietary polyactylene, Gymnasterkoreayne B, induces both intrinsic and extrinsic pathway of apoptosis through the signaling molecules revealed by secretome profiling (LB183)
Authors: Song Dae‐Geun , Kang Kyungsu , Lee Eun Ha , Jung Sang Hoon , Pan Cheol‐Ho , Nho Chu Won
Published: June 2021

Gymnasterkoreayne B (GKB) is a natural polyacetylene isolated from Gymnaster koraiensis. The antiproliferative activity of GKB has been reported in various human cancer cells but its mechanism is still unknown. Our objectivity of the study was to investigate the mechanism of the antiproliferative activity of GKB in HCT116 cancer cell and to search any possible candidates that may involve in the cell death signaling. In this study, we confirmed that GKB has induced apoptosis in HCT116 cells, inferentially from the increment of the sub G0/G1 content (%) and the externalization of phosphatidylserine, and the apoptotic morphological changes. We also verified that the antiproliferative activity of GKB was not necroptosis, by the cell death inhibitor assay using z‐VAD‐fmk and necrostatin. In addition, by the mitochondrial membrane potential assay and flow‐cytometric analysis, we elucidated that GKB treatment resulted in ROS accumulation, leading to the activation of both intrinsic and extrinsic apoptotic pathway. To investigate the effect of GKB on the secretome profile, we compared protein levels in the media of GKB‐ or vehicle‐treated HCT116 cells using 1D‐SDS‐PAGE and LC‐MS/MS analysis. Total of 1804 proteins were identified and their expression levels were determined by using spectral counting method. As a result, 70 proteins were down regulated (< 0.5 fold) and 56 proteins were up regulated (> 2.0 fold). Among these, the expression levels of apoptosis‐related proteins, ADAM9, interleukin 18, and serpin B5 were validated by ELISA. In depth analysis regarding the gene ontology of the identified protein suggested that FN1, TGFB1, APP, SERPINE1, HSPD1, SOD1, TXN, and ACTN4 may also act as secretory signaling molecules that trigger GKB‐induced apoptosis. In summary, we have found the mechanism of GKB‐induced apoptosis of HCT116 and we have suggested novel proteins which might be involved in the apoptotic signaling.

Grant Funding Source: Supported by Korea Institute of Science and Technology Gangneung Institute intramural research grant (2Z04220)
Twist1 induces dissemination by activating an epithelial motility program that requires E‐cadherin (LB230)
Authors: Shamir Eliah , Pappalardo Elisa , Tran Phuoc , Bader Joel , Ewald Andrew
Published: June 2021

Cancer metastasis begins with dissemination of cells from an epithelial tumor. Each tumor contains many mutations and a heterogeneous cell population. As such, it has been difficult to isolate the molecular requirements for a single epithelial cell to disseminate. One posited mechanism for dissemination is activation of an epithelial to mesenchymal transition (EMT), in which repression of the cell adhesion gene E‐cadherin is considered the driving molecular event. We sought to test the sufficiency of single gene perturbations to induce dissemination in primary murine mammary epithelium. Deletion of E‐cadherin disrupted simple architecture and branching morphogenesis but rarely resulted in dissemination. In contrast, expression of the EMT transcription factor Twist1 induced rapid dissemination of cells into the extracellular matrix. Twist1 induced acute transcriptional changes in cell‐matrix adhesion genes but not in epithelial‐specific genes, including E‐cadherin. Surprisingly, knockdown of E‐cadherin inhibited Twist1‐induced single cell dissemination and promoted collective epithelial migration. We conclude that Twist1 activates an epithelial motility program without requiring a transition to mesenchymal cell fate. To date, it remains unclear why some Twist1+ cells disseminate while many remain in the epithelium. We next seek to test the sufficiency of Twist1 to induce dissemination in distinct mammary epithelial subpopulations. We anticipate that cell type‐specific differences may suggest which cells within a heterogeneous tumor have the highest metastatic potential.
Differential roles of Sirt1 in HIF‐1a and HIF‐2a mediated hypoxic responses (LB269)
Authors: Yoon Haejin
Published: June 2021

Hypoxia‐inducible factors 1α and 2α (HIF‐1α and HIF‐2α) determine cancer cell fate under hypoxia. Despite the similarities of their structures, HIF‐1α and HIF‐2α have distinct roles in cancer growth under hypoxia, that is, HIF‐1α induces growth arrest whereas HIF‐2α promotes cell growth. Recently, sirtuin 1 (SIRT1) was reported to fine‐tune cellular responses to hypoxia by deacetylating HIF‐1α and HIF‐2α. Yet, the roles of SIRT1 in HIF‐1α and HIF‐2α functions have been controversial. We here investigated the precise roles of SIRT1 in HIF‐1α and HIF‐2α regulations. Immunological analyses revealed that HIF‐1α K674 and HIF‐2α K741 are acetylated by PCAF and CBP, respectively, but are deacetylated commonly by SIRT1. In the Gal4 reporter systems, SIRT1 was found to repress HIF‐1α activity constantly in ten cancer cell‐lines but to regulate HIF‐2α activity cell type‐dependently. Moreover, SIRT1 determined cell growth under hypoxia depending on HIF‐1α and HIF‐2α. Under hypoxia, SIRT1 promoted proliferation of HepG2 cells, in which SIRT1 differentially regulates HIF‐1α and HIF‐2α. In contrast, such an effect of SIRT1 was not shown in HCT116 cells, in which SIRT1 inactivates both HIF‐1α and HIF‐2α, because conflicting actions of HIF‐1α and HIF‐2α on cell growth may be offset. Our results provide a better understanding of the roles of SIRT1 in HIF‐mediated hypoxic responses and also a basic concept for anticancer strategy targeting SIRT1.

Key words: Sirtuin 1; lysine acetylation, hypoxia; hypoxia‐inducible factors; transcriptional activity
Sinigrin Suppresses Adipocyte Differentiation through the MAPKs Pathway in 3T3‐L1 Preadipocytes (LB362)
Authors: Jang Yeon jeong , Lee Heewoen
Published: June 2021

Obesity is a major risk factor for various diseases such as type 2 diabetes, hypertension, cardiovascular diseases and certain types of cancer. In these days, anti‐obesity effects of natural products are of intense medical interest. In the present study, we examined the inhibitory effects of sinigrin, belongs to the family of glucosides found in some plants of the Brassicaceae family such as Brussels sprouts, broccoli, and the seeds of black mustard (Brassica nigra), on adipocyte differentiation in 3T3‐L1 cells. Sinigrin attenuated the accumulation of lipid droplets stained with Oil Red O. The expression of peroxisome proliferator‐activated receptor γ (PPARγ), a main transcription factor for adipocyte differentiation, was dose dependently decreased by sinigrin treatment. Also, the effects of sinigrin on the expression of CCAAT/enhancer‐binding protein β (C/EBPβ) and MAPK activation were investigated. The results revealed that sinigrin sequentially inhibited the phosphorylation of ERK, p38 and JNK. Moreover, sinigrin decreased the phosphorylation of C/EBPβ at Thr188, an essential step for transcriptional activation of C/EBPβ. Our results indicate that the inhibition of adipocyte differentiaion by sinigrin may be related to the regulation of the MAPKs pathway. Thus, sinigrin could be a novel therapeutic candidate for regulating adipocyte differentiation.
Lipoperoxidation in meats cooked in conventional and combined ovens (LB381)
Authors: Chaud Daniela , Razera Érika , Machado Alisson , Abreu Edeli
Published: June 2021

Introduction: The foods of animal origin have variable amounts of cholesterol in their composition. During thermal processing of these foods a fraction of cholesterol can be oxidized by heat and oxygen, producing cholesterol oxides that are byproducts that are harmful to human health and can express cytotoxic, atherogenic, mutagenic and carcinogenic effects.

Objective: To evaluate the formation of cholesterol oxides in meat cooked in conventional and combined ovens.

Methods: This cross‐sectional study evaluated the Lipoperoxidation in four types of meat (beef, poultry, fish and pork), which were cooked in conventional and combined ovens, performed in duplicate. The procedures for extraction and analysis followed the TBAR method.

Results: The product with higher Lipoperoxidation was the poultry, followed by fish, beef and pork, in both cooking procedures. In all preparations which used the combined oven the formation of cholesterol oxides was higher.

Conclusion: The cooking in the combined oven was thermal processing that generated higher Lipoperoxidation in the products analyzed.
Study on the antioxidant effect of Siraegi and Gondeure extracts by in vitro assay system (LB390)
Authors: Kang Myunghwa , Shin Kikyung , Kim Myojung , Song Hyeonhae
Published: June 2021

To elucidate the functionality of Siraegi and Gondeure, their were extracted by solvent and their were investigated in in vitro system for antioxidant effect. Gondeure were boiling for two hours. Half of their were freezed and half of their were dried under 50 ℃. 3 g of each sample was added to 80% ethanol 60 mL and extracted by sonification during 4hour at 40 ℃.The extracts were diluted to a concentration 2000 ppm, 1000 ppm, 500 ppm, 250 ppm, 100 ppm with 80% ethanol . Hydrogen radical scavenging activity of Siraegi extracts were 52.19 ± 0.2253mg/ml in freezed Siraegi, and 30.29 ± 0.1860mg/ml in Gondeur. Phenolic acid compounds were 1.52 ± 0.0013mg/ml in freezed Siraegi, and 1.28 ± 0.0092mg/ml in hot air dried Siraegi. The reducing power was 2.12 ± 0.0148mg/ml in hot air dried Gondeure, the lowest in freezed Siraegi. Antioxidant capacity of Gondeure than the siraegi was good. Pre‐processing the activity of freeze‐dried samples than samples dried at 50 ℃ showed good activity.

Grant Funding Source: No
Evaluation of the anticancer activity of methionine methylsulfonium chloride: the involvement of SAPK/JNK activation (LB393)
Authors: Kim Gun‐Hee
Published: June 2021

The aim of the present study was to investigate the anticancer activity and mechanism of action of methanol extract from Artemisia princeps var. orientalis (APME). First, the antiproliferative activity of APME against T47D cells was at various concentrations (5‐200 μg/mL) and treatment times (24‐72 h) determined by MTT assay. APME significantly inhibited cell proliferation in a dose‐dependent manner, regardless of treatment times. Under the same condition, cytotoxicity (LDH release) of APME was only appeared at 72 h. In addition, treatment with the IC50 concentration of APME for 24 h caused cell cycle arrest and apoptosis. APME arrested cells in both G1 and G2/M phases of the cell cycle, a response that was related to the induction of CDK1, cyclin B, p21Cip1 expression, and repression of CDK2 and cyclin A. The induction of p21Cip1 by APME caused activation of p53 and modulation of its downstream targets Bcl‐2 and Bax. Subsequently, caspase‐8 and ‐9 were cleaved in cells treated with APME. Activation of these caspases in turn led to increased activation of caspase‐3. The results of this study suggest that APME produce anticancer effects in T47D cells through cell cycle arrest associated with the induction of p21Cip1 and apoptosis mediated by p53 and caspase‐depedent pathways.

Grant Funding Source: This work was supported by Priority Research Centers Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology(KNF‐2009‐0094017).
Increased capacity for glycogen turnover in the rat ventromedial hypothalamus (VMH) following hypoglycemia (LB449)
Authors: Mun Jonathan , Rhodes Justin , Beverly Lee
Published: June 2021

Recurrent hypoglycemia impairs the ability of the VMH to initiate a counterregulatory response and contributes to hypoglycemia‐associated autonomic failure (HAAF). We hypothesize that mobilization of VMH glycogen during and following hypoglycemia interferes with local glucose sensing and that genes associated with increased glycogen turnover will be upregulated after single and recurrent episodes of hypoglycemia. VMH were dissected from rats (N=23) 6 h after a single episode of insulin‐induced hypoglycemia (IIH) (acute), 24 h after the third of 3 recurrent episodes of IIH (recurrent), and saline‐treated euglycemia (control). RNA was extracted for RNA sequencing and real time‐PCR (RT‐PCR) analysis for glucose metabolism genes. One‐way ANOVA identified genes with statistically significant expression changes between treatments, principal component (PC) analysis of significantly different genes separated data points by treatment group, and genes significantly correlated with the PCs were identified. Glycogen synthase, phosphorylase kinase, glycogen phosphorylase, and UDP‐glucose pyrophosphorylase were significantly increased after acute hypoglycemia compared to control in the RT‐PCR analysis. Glycogen phosphorylase was upregulated in the acute treatment groups from both RNAseq and RT‐PCR compared to control and showed sustained elevation in the recurrent treatment from RT‐PCR analysis. These data provide evidence supporting the hypothesis that glycogen metabolism contributes to impaired glucose counterregulation and identify genes that are associated with novel hypotheses for future exploration. Supported by DK082609

Grant Funding Source: DK082609
Virus replication and cytokine production in primary alveolar macrophages and airway epithelial cells induced by emerging canine H3N8 influenza virus (LB510)
Authors: Castleman William , Hancock Calvin
Published: June 2021

Canine H3N8 influenza virus was first recognized as a serious infectious pulmonary disease in racing greyhound dogs in 2004. Disease resulted from direct transmission of commonly circulating equine H3N8 virus to dogs.

We hypothesized that transmission of equine influenza virus to dogs was characterized by mutations in one or more genes that allowed the virus to replicate more efficiently in dog airway epithelial cells and/or alveolar macrophages and induce higher levels of proinflammatory cytokines. Primary alveolar macrophages and airway epithelial cells were isolated from dogs and inoculated with emergent and endemic canine H3N8 influenza viruses A/canine/Florida /43/2004 (canine/FL/04), canine/FL/05, canine/PA/09 as well as early and more recent equine viruses equine/KY/91, equine/NY/99 and equine/OH/03.

Cytokines responses were measured by multiplex protein assay and/or quantitative PCR. Emergent and endemic canine viruses replicated to higher titers in dog macrophages than did the equine viruses from 1999 and 2003. Only endemic canine viruses from 2005 and 2009 replicated to a higher titer in isolated dog airway epithelial cells than did equine viruses from 1999 and 2005. Induction of TNF‐alpha in macrophages by equine and canine viruses was highly variable among macrophage isolates from different dogs. No significant differences were measured in epithelial cell production of IL‐8 in response to canine or equine H3N8 virus inoculation. Although small differences in productive virus replication were detected for canine influenza virus isolates compared to equine influenza virus isolates in macrophages and epithelial cells, differences in cytokine production were not detected.

Grant Funding Source: Supported by NIH AI101625
Expedited RPE‐derivation from human embryonic stem cells by partial PARP‐1 inhibition (LB55)
Authors: Pennington Britney , Clegg Dennis
Published: June 2021

The death of the retinal pigmented epithelium (RPE) induces the leading cause of blindness in the elderly, a disease called age‐related macular degeneration (AMD). Human embryonic stem cells (hESCs) have the potential to generate a limitless source of RPE for cellular therapies, but spontaneous differentiation methods require several months to produce mature cells. In 2013, a novel directed‐differentiation protocol described efficient derivation of RPE in 14 days using nicotinamide as a key‐inducing agent in the chemical cocktail. This study investigates the hypothesis that nicotinamide expedites RPE‐derivation by inhibiting poly(ADP‐ribose) polymerase‐1 (PARP‐1) during the neuralization component of differentiation. 3‐aminobenzamide (3‐ABA), a non‐hydrolyzable nicotinamide analogue and PARP‐1 inhibitor, was tested in place of nicotinamide. Gene expression analysis by qPCR, morphological assessment, and pigment quantification demonstrate that 3‐ABA affects differentiation during the first 4 days of differentiation, ultimately yielding cells with similar, yet attenuated, levels of RPE‐marker gene expression compared to controls. This suggests that nicotinamide contributes to RPE differentiation from hESCs by partially inhibiting PARP‐1. Since other mechanisms are likely involved, future investigation aims to identify additional pathways contributing to this efficient differentiation of RPE cells, which will be important for generating cells for therapeutic applications.

Grant Funding Source: Supported by Sigma Xi, Fight For Sight, and the California Institute for Regenerative Medicine
Sex differences in the expression of MOR in areas critical for fear conditioning and extinction learning (LB557)
Authors: Morales Silva Roberto , Perez‐Torres Emily , Santini Edwin , Torres‐Reveron Annelyn
Published: June 2021

The mu opioid receptor (MOR) has a significant role in fear conditioning and extinction learning. Communication between amygdala, periaqueductal gray (PAG) and medial prefrontal cortex (mPFC) regulates fear conditioning and extinction. Fear conditioned female rats during the metaestrus (M, low hormones) stage of the estrous cycle, showed impaired extinction while rats during the proestrus (P, high hormones) stage showed normal extinction learning. We aim to elucidate how opioid modulators alter MOR expression during fear conditioning and extinction in both sexes. Female rats on M or P stages and males rats underwent auditory fear conditioning and immediately received MOR agonist morphine, or antagonist naloxone or saline. The next day rats received extinction trials. Western blots were used to measure MOR expression. In the PAG, female rats showed no change with either treatment. In male rats morphine produced an increase in MOR expression while naloxone had an opposite effect. In the PFC, morphine during P decreased MOR expression while naloxone had no effect. Increased MOR expression was observed in the amygdala of male rats after either morphine or naloxone treatments, while in females an increase in MOR was only observed during P. Our data portraits sex differences in MOR expression in the fear circuitry, which might be responsible for the persistence of fear responses during low ovarian hormone stages.

Grant Funding Source: Supported by Molecular and Genomics core RR003050, Behavioral Core Facilities MD007579 , HPD335698 Nova Southeastern University to Edwin Santini and Dinah Ramos‐Ortolaza
Screening for and Identification of Novel Glucarpidase Producing Bacteria: Cloning and molecular characterisation of novel enzymes involved in ADEPT for cancer treatment (LB581)
Authors: Alqahtani Alanood , Alyafei Afrah , Abdallah Fatma , Latiff Aishah , Groves Mathew , Domling Alex , Goda Sayed
Published: June 2021

Antibody Directed Enzyme Prodrug Therapy (ADEPT) is a novel therapy which has already been implemented in cancer therapy to solve the problem of drug resistance and lack of tumor selectivity. Repeated cycles of (ADEPT) and the use of glucarpidase in detoxification of cytotoxic methotrexate (MTX) are desirable but are hindered by the induced human antibody response to the glucarpidase.

Novel glucarpidases with unrecognized epitopes may increase the prospect of repeated cycles of ADEPT for effective cancer therapy and could also be used to remove excess MXT.

The aim of this study was to isolate and characterize new glucarpidase (former name: carboxypeptidase G2) producing bacteria in order to clone and overexpress the cognate genes for Antibody Directed Enzyme Prodrug Therapy (ADEPT), and for detoxification of methotrexate (MXT) used for cancer treatment.

Over five hundred soil samples were collected from different fields in Egypt and Qatar where folate rich fruit and vegetables are routinely cultivated. Screening of these samples was performed using folate as the sole carbon and/or nitrogen source. Several molecular techniques including 16S rRNA were used to characterize the isolated strains.

The work led to the isolation and characterization of four new glucarpidase producing strains, The novel native enzymes have been assayed using Methotrexate as a substrate, and characterised.

We successfully cloned and molecular characterized a novel glucarpidase from one of the isolated strains which could be used in the ADEPT techniques for cancer treatment that could overcome the immune response problems.

Grant Funding Source: Qatar National research fund, NPRP 6 ‐ 724 ‐ 3 ‐ 183