bluuebunny/crossref_metadata_2025 huggingface dataset:
Bioactive Fungal Screening to find Efficient Biomass‐Degrading Enzymes
Authors: Vazquez Uriel Adan , Conrad Dale , Ma Hongyan , Woodard Toni , Wu Si
Published: June 2021

Traditional fuels such as coal and gasoline are limited and can produce harmful effects to the environment. Bioethanol from biomass (i.e., plant‐based materials) degradation is a sustainable and renewable form of fuel that aims to reduce these effects. The fungal secretome, known as a pool of biomass degrading enzymes, can be utilized to discover industrial enzyme candidates useful for biofuel production. The overall purpose of our research is to pre‐screen a pool of fungi collected from the Great Lakes, select candidates that can break down biomass (i.e., cellulose) efficiently, and characterize the biomass degrading enzymes. In this study, we first selected seven different fungal species, and grew them onto carboxymethyl cellulose (CMC) plates. A specific dye called Congo red was used to monitor the cellulose activity after either a five‐day or twelve‐day growth period. A clear zone present around the central fungal growth indicated the degradation of the cellulose in the culture medium. Fungal activity was measured using a ratio of enzyme activity (outer ring) and the fungal growth (central circle). The enzyme activities of these strains were compared to Aspergillus niger, a known industrial fungal strain. Overall, there were four different species that demonstrated higher cellulase activity in comparison to A.niger in the CMC assay. Additionally, the secretome and cell lysate of A. niger was prepared to determine the source of the biomass‐degrading enzymes through an enzymatic activity assay. Preparation of the secretome and lysate were obtained through ultra filtration and bead beating, respectively. Both methods resulted in acquiring a highly concentrated solution of each protein samples. It was determined that the biomass‐degrading enzymes were secreted from the fungal cells rather than contained within the cell. Future directions include secretome preparation of the four identified fungal strains to verify their biomass hydrolysis capabilities.

Support or Funding Information

This work was supported NSF award 1359457 to Dr. Si Wu.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sulfur respiration in A. fulgidus? Kinetics and Structure of an A. fulgidus NAD‐dependent Poly/Persulfide Reductase.
Authors: Shabdar Sherwin , Castineiras Ana , Desir Neyissa , Crane EJ , Sazinsky Matthew
Published: June 2021

PNDORs are a large class of homodimeric NADH and FAD‐dependent enzymes that generally function to reduce small molecule substrates at a catalytic cysteine positioned at the si face of the isoalloxazine ring. The substrate preference and reactivity vary greatly depending on surrounding catalytic residues and additional structural elements. Two closely related PNDORs have been found to have in vitro S0 reducing activities and have been correlated with in vivo S0 reduction – CoADR and Npsr. Both enzymes catalyze the general reaction of S80, Sn2− or CoA‐SnH + NAD(P)H‐> S2− + NAD(P)+ + CoA‐SH. The most obvious structural difference between the two enzymes is the addition of a rhodanase‐like domain on the Npsr which contains a surface‐located cysteine that we have shown to be the initial sulfur‐accepting site. CoADR is widely distributed throughout the thermophilic and hyperthermophilic archaea, while the Npsr is not found in the archaea, with the exception of an Npsr homologue found in the sulfate‐reducing thermophile Archaeoglobus fulgidus. Unlike the other CoADR and Npsr‐containing organisms, A. fulgidus cannot respire S0, and is actually inhibited by S0. Sequence homology suggests that this A. fulgidus Npsr should be a per/polysulfide reductase, however, the lack of a sulfur‐reducing phenotype suggests 1) the catalytic function of this enzyme is changed, and it fulfills a new function, or 2) the polysulfide reductase activity of the enzyme in vivo is not sufficient to confer the ability to respire S0 in the context of the A. fulgidus cell, and the enzyme's persulfide/polysulfide reductase activity fulfills a different role, perhaps acting in an antioxidant or detoxification reaction. To differentiate between these two possibilities and better understand archael sulfur respiration processes here we report the kinetics, substrate preferences and structure A. fulgidus Npsr.

Support or Funding Information

This material is based upon work supported by the National Science Foundation under Grant No. 1518306

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biosynthesis of nonproteinogenic amino acids oxyvinylglycines
Authors: Li Bo , Patteson Jon B. , Dunn Zachary D.
Published: June 2021

Nonproteinogenic amino acids (NAAs) are well known for their potent biological activities and unique chemical functionalities that are not encoded in proteins. NAAs are produced across all kingdoms of life, and many NAAs have broad applications in agriculture, nutrition, and medicine. The biosynthetic chemistries for many NAAs, however, remain largely unknown. I will discuss our recent results on reconstituting the biosynthesis of a class of NAAs called oxyvinylglycines, which exist as free amino acids and act as antimetabolites. Our work has uncovered unique enzymatic transformations involved in the biosynthesis of oxyvinylglycines. Understanding the biosynthetic chemistry of NAAs could facilitate the discovery of new enzyme catalysts and engineered biosynthesis of NAA‐containing natural products.

Support or Funding Information

This work is supported by National Institutes of Health (R00 GM099904), National Science Foundation (CHE1654678), the Rita Allen Foundation, and the University of North Carolina at Chapel Hill.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Computational Design and Simulation of a Cyclized Dimeric Multipass Transmembrane Protein
Authors: Aldana‐Mendoza Jonathan Agust , DeGrado William Agust
Published: June 2021

Transmembrane (TM) proteins play critical roles in cell surface markers, receptors, and can serve as channels for molecular transport. Therefore characterizing this structural class of proteins is important for understanding a wide variety of cellular functions. With the influx of solved structures of alpha helical TM proteins, we are better equipped to address the unique structural characteristics of these proteins. We hypothesize that a de novo design and synthesis of a tm protein cold be carried out with accurate atomic level prediction of its tertiary structure. We based our design on the characterized GX6G motif of alpha‐helical proteins; to set parameters for searching naturally occurring alpha‐helices in the PDB. Then using these naturally occurring proteins to analyze any sequence preference given the alpha‐helical structure. We plan on validating our design and simulation through the synthesis of this peptide using solid phase Fmoc synthesis. Structure analysis will be performed on the product through circular dichroism, infrared/vibrational, and NMR spectroscopy. The synthesis and accurate prediction of this alpha‐helical tm protein, will demonstrate our capacity to predict structures of tm proteins that contain the sequence/structural motifs through in cooperation in a rational design. Enabling us to rationally design tm proteins, or small molecules that target tm proteins, and therefore have powerful therapeutic potential.

Support or Funding Information

NIH Grant # GM 49001, CSU‐LSAMP is supported by the National Science Foundation under Grant # HRD‐1302873 and the CSU Office of the Chancellor

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Computational Design Of A Mimic Against Neurodegenerative Diseases Based On VEGF‐D
Authors: Mason Christopher , Bibi Najma , Zahran Mai
Published: June 2021

The basis of this research is the use of a computational approach to design drugs based on the tertiary structure of the protein VEGF‐D. Vascular Endothelial Growth Factor‐D has been discovered to have a profound role in neuronal growth in which it demonstrates its ability to repair memory and improve cognitive functions. This discovery of VEGF‐D's new function has fascinated researchers as it shows promising results of being a possible cure against neurodegenerative diseases such as Alzheimer's however, its inability to bypass the blood brain barrier has intrigued our collaborators to develop a set of peptides that may induce the same reaction as VEGF‐D. The purpose of this research is to analyze the structural and functional details of VEGF‐D complexed to its receptor VEGFR‐3 at atomic level, design smaller drugs that have the potential to act as VEGF‐D, and identify the best modeled peptide that interacts most favorably with the receptor to present to our collaborators for experimental testing. This work involves the designing of 14 small peptides for which molecular dynamic simulations were performed to investigate the behavior of the peptides along the trajectory. Information obtained from dynamics simulation denote peptide 56 as the most suitable candidate to induce the same reaction as VEGF‐D.

Support or Funding Information

New York College Of Technology, NSF

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determination of Iron in Biological Samples using Sector‐Field vs. Quadrupole Inductively Coupled Plasma Mass Spectrometry
Authors: AL‐Ogaidi Hussein A. , Ketterer Michael E. , Ragan Emily J.
Published: June 2021

Inductively coupled plasma mass spectrometry (ICPMS) has become the preferred technique for trace and ultra‐trace determination of elemental concentrations in a wide variety of sample matrices. In our studies of iron (Fe) uptake and metabolism in insect cells, we evaluate different ICPMS procedures to measure pg – ng quantities of Fe in whole insects and Drosophila melanogaster S2 cells (Sg4 isolate); samples are digested and mineralized by heating with nitric acid (HNO3) at 90°C in fluoropolymer containers. The determination of Fe by ICPMS is well‐known to be affected by interferences from polyatomic ions; e.g., the species 40Ar16O+ forms in the plasma and directly impacts the most abundant 56Fe isotope. We evaluate two ICPMS approaches for performing reliable low‐level Fe determinations: A) quadrupole ICPMS with a collision cell system for attenuation of polyatomic ions; and B) a sector field ICPMS system capable of producing high resolving powers (m/Δm ~ 10,000). The latter system is easily capable of physically separating the 56Fe+ peak (55.94 amu) from interfering 40Ar16O+ (55.96 amu), and can generate solution detection limits of ~ 0.01 mg L−1, which is far superior to the ~ 1 mg L−1 detection limits attainable by quadrupole ICPMS with a collision cell in the kinetic energy discrimination mode. We describe the relative merits of using internal standardization with 59Co vs. isotope dilution with a 57Fe‐enriched spike solution. Standard operating procedures for the determination of Fe using SF‐ICPMS will be discussed.

Support or Funding Information

NSF grant IOS‐1656407

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Large Gold Nanorods Affect Glutathione but not K+ Metabolism in Human Red Blood Cells
Authors: Alla Praveen K. , Ihezurike Nedu C. , Lauf Peter K. , de la Zerda Adam , SoRelle Elliot D. , Pavel‐Sizemore Ioana E. , Yaklic Jerome C. , Adragna Norma C.
Published: June 2021

With the advent of nanotechnology, the potential applications of nano‐scale gold in medicine have expanded substantially. Gold therapeutic and toxic properties have been widely studied, however little is known regarding gold's contribution to oxidative stress in red blood cells (RBCs). This is especially true for recently‐demonstrated large gold nanorods (LGNRs) (Langmuir 2015, 31, 12339–12347), which possess unmatched theranostic capabilities. Here, we report on the effect of LGNRs at the level of cellular function, and particularly on glutathione (GSH) concentration and ion transport in human (H) RBCs. Gold has been hypothesized to effectively react with RBCs sulfhydryl groups, thereby causing depletion of the antioxidant reserve represented by GSH and its pathway, through which GNRs induced cellular toxicity. In instances of acute exposure to gold, GSH is depleted and GSH synthesis is enhanced. This study examined the effect of LGNRs on the GSH concentration and K+ metabolism in cord and adult HRBCs. Our findings indicate that LGNRs induced changes in RBC GSH concentration in dose (3.3–40.0 μg/ml)‐ and time (15–60 min)‐dependent manners without affecting RBC K+ metabolism. Furthermore, there was inter‐individual variability in the GSH concentrations of both cord and adult RBCs, whereas there was no statistically significant difference between cord and adult RBCs. This study also revealed that LGNRs behaved either like oxidants, antioxidants or had no effect on GSH concentration in both cord and adult RBCs, which may be partly attributed to the initial state of oxidation of the blood. Moreover, the effect of LGNRs in dose‐response studies had a biphasic effect on GSH concentration, i.e. both behaving as oxidants and antioxidants as a function of their concentration in RBCs of the same donor, and between subjects at a given concentration. In conclusion, the unique properties of LGNRs could be further enhanced in a precision medicine setting.

Support or Funding Information

Supported in part by the National Science Foundation and by the Wright State University Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Liver Protein Comparisons of Warm‐adapted Versus Cold‐adapted Populations of Threespine Sticklebacks (Gasterosteus aculeatus)
Authors: Levitan Bryn Bo , Gómez‐Jiménez Silvia , Li Johnathon , Kültz Dietmar
Published: June 2021

This study investigated the molecular phenotypes of a warm‐adapted population of threespine sticklebacks (Gasterosteus aculeatus). We created a liver protein assay consisting of 1,016 proteins with Skyline (MacCoss Lab) from four populations (N=24) using data dependent acquisition (DDA) shotgun proteomics. This assay will be used to perform targeted proteomics on future comparisons, which will allow for more precise identification and quantification of the targeted proteins. A population of sticklebacks from Baja California, Mexico is surviving and breeding at temperatures near 30 degrees Celsius, which is close to the upper thermal limit for this species. Understanding what this population is doing at the molecular level to function at these high temperatures may give insight into mechanisms that have allowed for their survival, as well as the potential for other populations to adapt to higher temperatures. Livers from sticklebacks of the Baja California population were then compared to three cold‐adapted populations using gel‐free quantitative proteomics by liquid chromatography‐mass spectrometry (LC‐MS/MS). A resident marine population (Bodega Harbor, CA), a freshwater population (Lake Solano, CA), and an anadromous population (Anchorage, Alaska) were used in these comparisons. Six Baja California threespine sticklebacks and six fish from each of the other three groups were compared in four separate sets (N=96). Approximately 20 proteins were significantly more abundant in the Baja California population than the other three populations across all four sets. Our results suggest that certain molecular chaperones and redox/antioxidant proteins may contribute to the survival of the Baja California population. Our study lays the foundation for highly accurate quantification of protein differences in future comparisons using targeted proteomics. This study also highlights proteins that are consistently found at higher abundance in the warm‐adapted population and thus may be important for liver adaptation to warmer temperatures.

Support or Funding Information

Funded by NSF Grant IOS‐1355098.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Paper‐Based Breast Tumor Model Reveals New Insights into the Hypoxic Regulation of Estrogen Receptor Alpha
Authors: Whitman Nathan , Lin Zhi‐Wei , Kenney Rachael , Lockett Matthew Ryen
Published: June 2021

Nearly 75% of breast tumors express estrogen receptor alpha (ERα). These tumors are treated with endocrine therapies to block ERα‐directed transcription and halt tumor progression. However, acquired resistance to these therapies is common, and associated with a poor clinical prognosis. Hypoxia has been implicated in acquired endocrine resistance through loss of ERα expression based on studies conducted using 2D monolayer cultures. However, the impact of hypoxia on ERα expression and estrogen signaling has not been evaluated in 3D culture models, which recapitulate the cell‐cell and cell‐matrix interactions in vivo.

Here, we compare the impact of hypoxia on ERα expression and estrogen signaling between 2D monolayer cultures and 3D paper‐based cultures. Paper‐based cultures are an emerging 3D culture system that relies on paper scaffolds to generate tissue‐like constructs whose microenvironments can be controlled and measured. Using the ERα‐positive cell lines, T47D‐KBluc and MCF‐7, we have demonstrated that ERα levels in 3D paper‐based cultures are insensitive to hypoxia (1% O2), yet estrogen signaling decreases under hypoxic conditions. To understand this inconsistency with 2D cultures, we compare the role of hypoxia inducible factor 1α in regulating ERα and estrogen signaling, and compare ERα distribution and localization between the two culture formats.

Finally, we generate a breast tumor model by stacking individual scaffolds, which were seeded with ERα‐positive cell lines within a collagen matrix. We confirm and measure oxygen gradients within the tumor model using an oxygen‐sensitive thin film sensor, and evaluate the impacts of hypoxic tumor gradients on ERα and estrogen signaling. This work will provide new insights into the role of hypoxia in acquired endocrine resistance and generate a breast tumor model that will be further used study acquired endocrine resistance in a physiologically relevant in vitro tumor model.

Support or Funding Information

The project described was supported by the National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, through Grant Award Number UL1TR001111, along with Eli Lilly and Company's Young Investigator Award. ZWL was supported by the NC Louis Stokes Alliance for Minority Participation, NSF #1202467.

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 Role of the Vacuolar (H+)‐ATPase in Neuroblastoma Cell Differentiation induced by microRNA 506‐3p
Authors: Medrano Geraldo , Zhao Zhenze , Du Liqin
Published: June 2021

Neuroblastoma is one leading causes of cancer‐related childhood deaths. Neuroblastoma arises from the developing sympathetic nervous system where neural crest cells fail to complete the differentiation process. The failure in the differentiation process initiated the investigation into differentiation‐inducing agents. MicroRNA 506‐3p (miRNA‐506) was previously identified to be a potent tumor suppressor and induced neuroblastoma cell differentiation. A gene expression array determined that the ATP‐driven proton pump, vacuolar (H+)‐ATPase (V‐ATPase) is directly targeted by the miRNA‐506. However, the role of V‐ATPase in neuroblastoma has not been investigated. We recently found that by using siRNAs to knock down V‐ATPase expression we were able to reduce neuroblastoma proliferation and cell viability. These results warrant further investigation to determine the molecular mechanisms through which V‐ATPase helps mediate the effects of miRNA‐506 on neuroblastoma cells.

Support or Funding Information

The authors would like to thank the support for this project provided in part by the DoD, Texas State University, and South Texas Doctoral Bridge Program (GM102783)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FOXO Transcription Factors Rewire Metabolism in U87MG Glioblastoma Cells
Authors: Keniry Megan , Fanniel Victor , Martinez Eduardo , Sanchez Lilia , Vazquez Neftali , Lopez Alma , Cedillo Raechel , Respondek Christa , Gilkerson Robert , Innis‐Whitehouse Wendy , Scheunzel Erin
Published: June 2021

Evolutionarily‐conserved FOXO transcription factors reside in the nucleus of glioblastoma and basal breast cancer cells that harbor constitutive PI3K Pathway output. Nuclear localization of FOXO factors in this setting challenges canonical PI3K models, as this pathway typically induces FOXO localization to the cytoplasm. Simultaneous PI3K activation and nuclear FOXO have also been observed in a set of diffuse large B cell lymphomas and embryonic stem cells. To interrogate the function of nuclear‐localized FOXO transcription factors in glioblastoma and basal breast cancer cell lines, we have constructed genetic models using CRISPR Cas9 genome editing technology. Our evidence indicates that FOXO factors drive metabolic changes in these cancers. FOXO disruption mutants showed increased mitochondrial membrane potential by TMRE staining and subsequent flow cytometric analyses. FOXO factors promote PEPCK gene expression to likely impact carbon utilization. These findings suggest that the nuclear localization of FOXO transcription factors causes metabolic rewiring in glioblastoma and basal breast cancer.

Support or Funding Information

This work was supported by HHMI 52007568 (N.V.), USDA Step 2 2015‐38422‐24061(A.L., R.G,), USDA H.S.I. 2016‐38422‐25760 (M.K. and E.M), NIH 1SC3GM11666901 (R.G.), UTRGV College of Sciences (COS) Seed Grant (M.K.), and NSF Advance 1209210 (M.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.
Neurochemical Pathways Involved in A1 Adenosine Receptor Agonist‐Induced Hibernation in the Arctic Ground Squirrel (Urocitellus parryii)
Authors: Jenkins Mackenzie , Frare Carla , Drew Kelly
Published: June 2021

Hibernation is a phenomenon brought on by seasonal changes in phenotype. In the arctic ground squirrels (AGS), the effect of these seasonal changes is very evident. In late fall AGS decrease their metabolic demand which is followed by the entry into hibernation in winter. Previously the lab has shown that N6‐cyclohexyladenosine (CHA), an A1 adenosine receptor agonist, induces hibernation in AGS in a seasonally dependent manner. AGS treated with CHA in winter experience a decrease in the rate of oxygen consumption and temperature as seen during natural hibernation during winter. However, in summer CHA triggers a brief and transient metabolic suppression that does not lead to hibernation onset. CHA induces hibernation at a location within the CNS, but specific sites of action are unknown. Here we utilize cFos expression to identify neuronal cell groups differentially activated in summer and in winter after CHA.

AGS implanted with body temperature (Tb) transmitter, were treated with CHA or vehicle (0.5 mg/kg, IP). Metabolic rate was measured via open‐flow respirometry. AGS were perfused with 4% paraformaldehyde and brains were dissected and cryoprotected in 5%, 10%, 15%, 20%, 30% sucrose. We performed free‐floating immunohistochemistry on 40 μm brain slices in four experimental groups: winter CHA, winter control, summer CHA and summer control. We sectioned brains from eight AGS per group for a total of 32 animals and identified active nuclei with cFos immunoreactivity (mouse anti‐cFos 1:20,000, Millipore). Blinded analysis of cFos immunoreactive neurons was done using bright field microscopy and Metamorph software. Statistical analysis was performed on R.

CHA produced a hibernation‐like response measured as a decrease in metabolic rate and Tb in winter AGS but not in the summer. Two regions showed greater activation following CHA indicated by higher count of cFos+ neurons: the Nucleus Tractus Solitarius (NTS) and the Median Preoptic Nucleus (MnPO). The NTS has been described previously as a site of action for a CHA‐induced hibernation‐like response in rats. Our data confirmed a higher NTS activation in CHA treated animals compared to vehicle. The MnPO, known to regulate thermogenesis, showed higher activation in winter CHA compared to vehicle. In conclusion CHA‐induced hibernation correlates with a decrease in thermogenesis.

Support or Funding Information

Research was supported by NIH R03 NS081637, NSF IOS‐1258179, RL5GM118990 and Alaska INBRE P20GM103395.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Characterization of the TIL 1383I T Cell Receptor
Authors: Davancaze Lauren M. , Singh Nishant K. , Anderson Michael J. , Huyke Fernando A. , Nishimura Michael I. , Baker Brian M.
Published: June 2021

Using adoptive cell transfer to reintroduce engineered T cells into patients is an exciting new form of cancer immunotherapy. T cells play a central role in adaptive immunity by eliminating virus‐infected and tumor cells. T cell receptors (TCRs) facilitate this by recognizing antigenic peptides presented by major histocompatibility complexes (MHCs), triggering an immune response. While TCRs in gene‐modified T cells can be used for anti‐tumor clinical responses, clinical trials have shown adverse effects attributable to off‐target toxicity, which stems from the inherent cross‐reactivity of a TCR repertoire developed to engage the larger universe of potential peptide antigens. To understand and begin to address this challenge in immunotherapy, elucidation of TCR structural properties and biochemical characteristics is required.

TIL 1383I is a T cell receptor (TCR) that targets the human tyrosinase peptide (hTyr) overexpressed in primary and metastatic melanoma. A recent phase I clinical trial of TIL 1383I using TCR‐gene modified T cells to treat melanoma resulted in one case of durable cancer remission but other cases with partial to no responses. To help understand and possibly engineer improved variants of TIL 1383I, we recombinantly expressed TIL 1383I and crystallized it in complex with its binding partner hTyr presented by HLA‐A2. We then used X‐ray crystallography to solve the structure. The structure provides insight into the binding geometry and amino acid residues in the interface between TIL 1383I and hTyr/HLA‐A2. We have also used surface plasmon resonance to determine the TIL 1383I binding affinity. From this work, we aim to design TCR variants with the goal of improving affinity and specificity toward the hTyr antigen.

Support or Funding Information

Harper Cancer Research Institute, National Institutes of Health, National Science Foundation, American Cancer Society

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the regulation of intercellular trafficking in plants with varying levels of thioglucoside glucohydrolase mutants
Authors: Fernandez Jessica Cisneros , Hewezi Tarek , Burch‐Smith Tessa
Published: June 2021

Plants have evolved cell‐wall pores called plasmodesmata to allow direct communication between connected cells. Plasmodesmata facilitate the intercellular trafficking of nutrients, metabolites and signaling molecules including hormones, small RNAs, and proteins. In the Arabidopsis thaliana increased size exclusion limit 2 (ise2) mutant, there is increased intercellular trafficking and increased numbers of branched plasmodesmata. Similar changes have been observed in ISE2‐silenced Nicotiana benthamiana leaves. In an effort to identify proteins that interact with ISE2, a pull‐down assay with purified MBP‐ISE2 as bait followed by protein identification by mass spectrometry was performed. THIOGLUCOSIDE GLUCOHYDROLASE 2 (TGG2) emerged as a potential ISE2‐interacting protein. In a separate gene expression analysis, expression of TGG1 was significantly decreased in ise2 mutant embryos compared to wild‐type controls. TGGs are myrosinases, enzymes responsible for catalyzing the hydrolysis of glucosinolates to unstable aglycons, which are re‐arranged to form toxic defense compounds like isothiocyanates.

In A. thaliana, TGG1 and TGG2 are duplicate genes expressed in above‐ground tissues. To investigate the relationship between ISE2 and TGG1 and/or TGG2, we monitored expression of genes in the glucosinolate pathway in plants with varying levels of ISE2 expression. We also measured intercellular trafficking in mutants of the glucosinolate pathway. Preliminary data suggest that overexpression of TGG1 increased nematode susceptibility in two lines and two mutant lines, tgg1–1 and tgg1–2, showed no significant difference from the wild‐type. Further, glucosinolate levels and intercellular trafficking were measured in plants overexpressing myrosinase TGG1. The results suggest that glucosinolates could play a role in mediating ISE2's effect on intercellular trafficking, particularly during plant defense.

Support or Funding Information

TBS supported by National Science Foundation Award 1456761 and start‐up funds from the University of Tennessee

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Catalytic strategy and inhibition of the prokaryotic specific GTP cyclohydrolase IB
Authors: Paranagama Naduni , Bonnett Shilah , Alvarez Jonathan , Luthra Amit , Stec Boguslaw , Gustafson Andrew , Samaan George , Purse Byron , Iwata‐Reuyl Dirk , Swairjo Manal
Published: June 2021

GTP cyclohydrolase I catalyzes the first step in folic acid biosynthesis in bacteria and plants, biopterin biosynthesis in mammals, and the biosynthesis of 7‐deazaguanosine modified tRNA nucleosides in bacteria and archaea. The type IB GTP cyclohydrolase (GCYH‐IB) is a prokaryotic‐specific enzyme found in several pathogens. GCYH‐IB is structurally distinct from the canonical type IA GTP cyclohydrolase involved in biopterin biosynthesis in humans and animals, and thus is of interest as a potential antibacterial drug target. We report kinetic and inhibition data of Neisseria gonorrhoeae (Ng) GCYH‐IB, and two high‐resolution crystal structures of the enzyme; one in complex with the reaction intermediate analog and a competitive inhibitor 8‐oxo‐GTP, and one with a TRIS molecule bound in the active site which mimics another reaction intermediate. Comparison of these GCYH‐IB crystal structures with the type IA enzyme bound to 8‐oxo‐GTP, reveals an inverted binding mode of the inhibitor's ribosyl moiety. Taken together with site‐directed mutagenesis data, suggests that the two enzymes utilize different strategies for catalysis. Additionally, the inhibitor interacts with a conserved active site residue Cys149 which is S‐nitrosylated in the structures. This is the first structural characterization of a biologically S‐nitrosylated bacterial protein. Mutagenesis and biochemical analyses demonstrate that Cys149 is essential for the cyclohydrolase reaction, and S‐nitrosylation maintains enzyme activity, suggesting a potential role of the S‐nitrosothiol in catalysis. These structures and information were used to perform structure based drug design using a combination of in silico screening and de novo design methods to generate molecules that inhibit NgGCYH‐IB selectively over human GCYH‐IA. A few compounds named G1, G2 and G1‐triphosphate have been generated and currently being tested for inhibition.

Support or Funding Information

This project was supported by NSF grant CHE‐1309323 to D. Iwata‐Reuyl and M.A. Swairjo,NIH grant GM110588 to M.A. Swairjo and D. Iwata‐Reuyl, NIH grant GM70641 to D. Iwata‐Reuyl, andan intramural grant from Western University of Health Sciences to M.A. Swairjo.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
1‐Formyl‐7‐hydroxy‐6,7‐dihydro‐5H‐pyrrolizine (9‐CHO‐DHP) ‐ A Biologically Proximate Pyrrolic Metabolite of Carcinogenic Pyrrolizidine Alkaloids
Authors: Xia Qingsu , He Xiaobo , Lin Ge , Fu Peter
Published: June 2021

Pyrrolizidine alkaloids (PAs) are phytochemical constituents of more than 6000 plant species worldwide; approximately half of the PAs are hepatotoxic, genotoxic, and carcinogenic. Because of their wide exposure and carcinogenicity, the International Programme on Chemical Safety (IPCS) concluded that PAs are a threat to human health and safety. We recently determined that the metabolic activation pathway of PA‐induced liver tumor initiation is mediated by a set of four (±)‐6,7‐dihydro‐7‐hydroxy‐1‐hydroxymethyl‐5H‐pyrrolizine (DHP)‐derived DNA adducts, and proposed that these DHP‐DNA adducts are potential biomarkers of PA exposure and liver tumor initiation. To validate the generality of this metabolic activation pathway and DHP‐DNA adducts as biomarkers, it is important to identify the spectrum of reactive metabolites associated with this metabolic activation pathway. Segall et al. (Drug Metab. Disposit., 12, 68–71, 1984) reported that 1‐formyl‐7‐hydroxy‐6,7‐dihydro‐5H‐pyrrolizine (9‐CHO‐DHP) is a metabolite formed from the mouse liver microsomal metabolism of senecionine. In this present study, we examined the metabolism of six hepatocarcinogenic PAs (riddelliine, senecionine, monocrotaline, senkirkine, intermedine, and heliotrine) and one non‐carcinogenic PA (platphylline) by human, rat, and mouse liver microsomes. 9‐CHO‐DHP was found as a common metabolite from the metabolism of the six hepatotoxic PAs, but not from platphylline. Incubation of 9‐CHO‐DHP in HepG2 cells and A549 cells resulted in the formation of the same set of DHP‐DNA adducts, as assessed by both LC/MS SRM mode and selected ion monitoring analyses through comparison to synthetic standards. In the incubation medium of HepG2 cells, both DHP and 7‐cysteine‐DHP were identified. These metabolites have previously shown to be capable of binding to cellular DNA to produce DHP‐DNA adducts. Thus, when incubated with HepG2 cells, 9‐CHO‐DHP was metabolized to the reactive metabolites DHP and 7‐cysteine‐DHP, metabolites associated with the DHP‐DNA adduct formation. These results indicate that 9‐CHO‐DHP is a DNA proximate metabolite of hepatotoxic PAs and further support the generality of this metabolic activation pathway mediated by the formation of these DHP‐DNA adducts. [This abstract is not an official U.S. Food and Drug Administration (FDA) guidance or policy statement. No offcial support or endorsement by the U.S. FDA is intended or should be inferred. The authors declare no competing financial interest.]

Support or Funding Information

This research was supported in part by appointment (X.H.) to the Postgraduate Research Program at the NCTR administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the FDA.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interaction of Fads2 Genotype and Dietary Essential Fatty Acid Intake on Metabolic Risk in Mice
Authors: Linde Peter E. , Evans Amanda J. , Li Puma Lance C. , Whitaker Connor M. , Worker Caleb J. , Chicco Adam J.
Published: June 2021

Polyunsaturated fatty acids (PUFAs) play important roles in inflammation, cardiovascular, and metabolic function. However, controversy exists regarding effects of dietary PUFA intake on metabolic risk due to inconsistent results from studies in the literature. We hypothesized that some of this controversy results from variations in individuals' metabolism of dietary essential PUFAs, suggested by altered cardiometabolic risk profiles in populations with polymorphisms in a key PUFA metabolism gene, FADS2. To address this, we developed colonies of mice with global heterozygous knockout (low), wild‐type (WT; medium) or mild transgenic overexpression (high) of FADS2 expression. Mice were fed a standardized high‐fat (40% kcal) diet containing dietary essential PUFAs at ~7% kcals provided as mostly linoleic acid (18:2n6, LA; as corn oil; 57:1 n6:n3), α‐linolenic acid (18:3n3; as flax oil; 1:4 n6:n3) or a 50/50 blend of each providing 1:1 n6:n3 PUFAs. Effects on glucose tolerance were monitored over 4 months, followed by analysis of liver fat deposition and macrophage content. In mice with WT FADS2 expression, enrichment of a high‐fat diet with n6‐PUFA augmented glucose intolerance, weight gain, and hepatic inflammation; while n3‐PUFA tended to have the opposite effect. The same metabolic syndrome phenotype was augmented by high (transgenic) FADS2 overexpression and attenuated by low FADS2 (knockdown) expression compared to WT controls when dietary n3:n6 PUFA composition was balanced 1:1, demonstrating a potent influence of FADS2 expression on metabolic phenotype and hepatic inflammation. However, dietary imbalance of n3:n6 PUFA intake, either toward higher n3 or n6 PUFA intake, tended to mitigate the effect of higher or low FADS2 expression. Taken together, these results support a complex interaction of FADS2 expression and dietary PUFA intake on metabolic risk, prompting future study of FADS2 haplotypes as potentially relevant criteria for individualized dietary recommendations.

Support or Funding Information

This project is supported by AFRI Grant # 2014‐06594 from the USDA National Institute of Food and Agriculture.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Resuscitation Adjuncts on Indices of Inflammation and Oxidant Stress in Tissues from Swine Subjected to Traumatic Hemorrhage
Authors: Barr Johnny L. , Martini Wenjun Z. , Thomas Theo W. , Dubick Michael A.
Published: June 2021

Introduction

In the absence of blood products, pre‐hospitaluse of small volume adjuncts to enhance crystalloid resuscitation has beenproposed to reduce inflammation and improve outcomes in the bleeding traumapatient. However, the most effective drugs for this purpose remain underintense investigation. The present study investigated the effects of Poloxamer‐188(P‐188), a surfactant with membrane resealing activity andethynylestradiol‐3‐sulfate (EE‐3‐S), an estrogen compound, on indices ofinflammation and oxidant stress in tissues from swine subjected to traumatichemorrhage. Both compounds are suggested to have anti‐inflammatory andpotentially anti‐oxidant activity.

Methods

Thirty anesthetized and instrumented pigs weresubjected to a left leg femur fracture followed by hemorrhage of 55% of the estimatedblood volume and a 15 min shock period. Pigs were then randomized into 3 groups(n=10/group) and resuscitated with equal small total volumes of normal saline (NS;4ml/kg) alone or NS containing P188 (200 mg/kg) or EE‐3‐S (1 mg/kg). Animalswere continuously monitored under anesthesia for 6h or until death. At death oreuthanasia, samples of liver, kidney, lung, heart and jejunum were flash‐frozenand stored at −80°C until assayed for antioxidant status, antioxidantenzyme activities and select cytokines.

Results

Hemorrhage reduced mean arterial pressure to 54±6%of baseline levels (p<0.05). In all tissues, total antioxidant status, reduced glutathione and antioxidant enzyme activities were similar amonggroups. In lung, however, nitric oxidelevels were 28% lower in the P‐188 treated than NS group (p<0.05). In liver, levels of IL‐1β, TNF‐α and IL‐6 were 25–30% lower in the P‐188 and EE3‐S treatedthan NS resuscitated swine (p<0.05), whereas IL‐10 levels were similar amonggroups. No statistically significant differences in cytokine concentrationswere observed in kidney, lung, heart or jejunum among the groups.

Conclusions

These data suggest that in this swine model oftraumatic hemorrhage and small volume resuscitation with crystalloid containingP‐188 or EE3‐S, any anti‐inflammatory activity was only observed in the liver.

Support or Funding Information

US Army Institute of Surgical Research, San Antonio, TX 78234

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CB2 Receptor Role in Anxiety‐like Behavior Using Validated CB2‐KO Zebrafish
Authors: Acevedo‐Canabal Agnes , Colon‐Cruz Luis , Varshney Gaurav , Behra Martine , Yudowski Guillermo
Published: June 2021

More than 100 million people use Marijuana or a synthetic analog worldwide, but effects and side effects are still poorly understood. Most cannabinoids, like Δ9‐tetrahydrocannabinol (Δ9‐THC), the main active constituent of marijuana, modulate the mesocorticolimbic system through binding to two cannabinoid receptors (CBRs) which are present in all vertebrates. The cnr1 and cnr2 genes encode the cannabinoid receptor 1 (CB1) and 2 (CB2), respectively. While CB1 is the most abundant G protein‐coupled receptor (GPCR) in the central nervous system (CNS), CB2 was thought to be primarily expressed in immune cells where it plays a major role in regulating the inflammatory response. However, there is increase evidence for expression and biological roles of CB2 receptor in the CNS. Thus, the goal of our experiments was to identify such role(s) by examining “anxiety‐like” behaviors in larval zebrafish. First, we established CB2‐knockout (CB2‐KO) animals using the CRISPR/Cas9 genome‐editing tool. An allele carrying an early 2bp deletion (cnr22Δ) in the coding sequence resulted in a loss of function mutation that we validated via Western Blots showing that the CB2 protein was absent in the adult homozygote brains (cnr2−/−2D). Next, we used a larval behavioral assay based on the photo‐dependent swimming response (PDR) and measured three anxiety‐like parameters (1) hyper/hypo activity, (2) Center avoidance and (3) photo‐stimulated startle response (PSSR) in wild type and CB2‐KO larvae. We found that all of those “anxiety‐like” behaviors were affected in the animals lacking CB2, thus clearly establishing this receptor as an important modulator of those behaviors.

Support or Funding Information

National Institute of Drug Abuse (R01.DA037924) and the National Science Foundation (HRD.1137725)‐Puerto Rico Center for Environmental Neuroscience (PRCEN).

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 Origin of the Avian Head as told by Transformations in the Fossil Record and Embryonic Development
Authors: Bhullar Bhart‐Anjan S.
Published: June 2021

The head of birds is immediately and profoundly distinguishable from those of other major vertebrate clades, owing to a bewildering number of derived features including an expanded brain and eyes, a shortening of the maxillary face, and an expansion of the premaxillary region at the tip of the snout into the characteristic avian beak. Considered separately, multitudes of individual transformations are required to bridge the gap between an ancestral reptilian cranium and a bird skull. However, in our work, we have found that large‐scale cranial evolution operates in such a way that multiple transformations have a single underlying developmental cause, resulting in character nonindependence and a simpler and more elegant evolutionary story than has previously been assumed. Foremost, we find that the avialan skull is fundamentally a juvenilized or paedomorphic version of the ancestral dinosaurian skull, and that many if not most of the iconic features of birds can be explained using a simple growth or scaling mechanism. In addition, the beak and skull roof — the entire modern avian feeding system, which features a great deal of mobility or kinesis — is patterned by one or a few early developmental influences. Finally, the highly modified avian skull roof is primarily influenced by the transformed brain, and indeed evidence suggests that a tight linkage between these structures is ancestral for Osteichthyes. We also found indications that the expanding brain restricted the size of the jaw‐closing musculature. Remarkably, the fossil record supports many of these hypotheses, showing clear signals of juvenilization and correlated shifts in brain and skull, and also showing simultaneous transformation of the rostral beak and the palate as suggested by our developmental work. We find particular support for our hypotheses using new material of the most important fossil taxa demonstrating the “modernization” of the avian skull, the Cretaceous toothed stem‐birds Ichthyornis and Hesperornis. However, this material simultaneously demonstrates that the avian adductor chamber was, in at least some taxa, remarkably plesiomorphic even after the expansion of the brain, falsifying our hypotheses of a strict causal relationship between brain expansion and adductor diminution. Thus, despite the importance of developmental data in demonstrating the ways in which character transformations are interrelated during major evolutionary transitions, the fossil record is always the final arbiter of the merit of any large‐scale evolutionary hypothesis.

Support or Funding Information

Funding provided by Yale University, the Yale Peabody Museum of Natural History, the National Science Foundation, and the Human Frontier Science Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.